Patent Publication Number: US-2021180007-A1

Title: Engineered microorganisms for the deconstruction of polymers

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. § 119 to PCT/US19/32480 filed on 15 May 2019, which claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62/671,477 filed on 15 May 2018, the contents of which are hereby incorporated in their entirety. 
    
    
     CONTRACTUAL ORIGIN 
     The United States Government has rights in this disclosure under Contract No. DE-AC36-08G028308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy as filed herewith was originally created on May 15, 2019. The ASCII copy as filed herewith is named NREL 18-76_ST25.txt, is 70 kilobytes in size and is submitted with the instant application. 
     BACKGROUND 
     Poly (ethylene terephthalate) (PET) is one of the most abundant manmade synthetic polyesters. Crystalline PET is being widely used for production of single-use beverage bottles, clothing, packaging, and carpeting materials. PET resistance to biodegradation due to limited accessibility to ester linkage, and disposal of PET products into the environment pose a serious threat to biosphere, particularly to marine environment. PET can be chemically recycled; however, the extra costs in chemical recycling are not justified when converting PET back to PET. Thus, there remains a need for alternative strategies for recycling/recovering/reusing PET. 
     SUMMARY 
     In an aspect disclosed herein is a genetically modified organism comprising:
         an exogenous gene addition, wherein:   the exogenous gene addition encodes functional enzymes comprising a PETase and a MHETase, and   the genetically modified organism is capable of metabolizing poly (ethylene terephthalate) (PET) to produce PET deconstruction products. In an embodiment, the genetically modified organism has an exogenous gene is from  Ideonella sakaiensis.  In another embodiment, the genetically modified organism has an exogenous gene is codon optimized. In another embodiment, the genetically modified organism has an exogenous gene is incorporated into the genome of the genetically modified organism. In another embodiment, the genetically modified organism has an exogenous gene addition further comprises genes encoding a secretion signal peptide. In another embodiment, the genetically modified organism has a genetically modified organism is a species of  Pseudomonas.  In another embodiment, the genetically modified organism is the species is  Pseudomonas putida.  In another embodiment, the genetically modified organism has PET deconstruction products comprise at least one of bis(2-Hydroxyethyl) terephthalate, mono-(2-hydroxyethyl) terephthalate, terephthalate, ethylene glycol, β-ketoadipate, or muconate. In another embodiment, the method comprising contacting poly (ethylene terephthalate) (PET) with the genetically modified organisms of claims  1  to produce PET deconstruction products. In another embodiment, the method of claim  9 , wherein the contacting is performed in minimal salt medium. In another embodiment, a genetically modified organism comprising:   an exogenous gene addition, wherein:   the exogenous gene addition encodes functional enzymes comprising a PETase and a MHETase, and   the genetically modified organism is capable of metabolizing poly (ethylene terephthalate) (PET) to produce PET deconstruction products; and   wherein said genetically modified organism further comprises heterologous TPA transporters. In another embodiment, the genetically modified organism further comprising catabolic gene clusters I or II. In another embodiment, the genetically modified organism wherein the catabolic gene clusters I or II are from  Comamonas  sp. E6. In another embodiment, the genetically modified organism is capable of using TPA as a sole carbon source. In another embodiment, the genetically modified organism is capable of metabolizing TPA at about 0.05 g L −1  h −1 . In another embodiment, the genetically modified organism is lacking a pcaIJ gene. In another embodiment, the genetically modified organism is capable of metabolizing TPA to β-ketoadipate. In another embodiment, the genetically modified organism is a species of  Pseudomonas.  In another embodiment, the genetically modified organism the exogenous gene is from  Ideonella sakaiensis.  In another embodiment, the genetically modified organism has a PET deconstruction products that comprise at least one of bis(2-Hydroxyethyl) terephthalate, mono-(2-hydroxyethyl) terephthalate, terephthalate, ethylene glycol, β-ketoadipate, or muconate.       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  depicts: Panel 1A illustrates bright field microscopic observation of the strain expressing PETase with GFP tag; Panel 1B illustrates microscopic observation of GFP signal of the strain expressing PETase with GFP tag; Panel 1C illustrates GFP signal of the supernatant of wild-type strain and the strain expressing GFP tagged PETase; Panel 1D illustrates immunoprecipitation of GFP tagged PETase with GFP specific GFP-Trap® (ChromoTek GmbH, Planegg-Martinsried, Germany); and Panel 1E illustrates a microscopic image of PET particle incubated with the strain expressing GFP tagged PETase. 
         FIG. 2  depicts degradation results of PET by LJ041 (Panel 2A) integrated gene cassette (Panel 2B) visual observation of biofilm of 1141 on PET film (arrow) (Panel 2C) fragmenting PET by LJ041 (Panel 2D) SEM observation of PET particles cultured with KT2440, after 5 days of incubation (Panel 2E) SEM observation of PET cultured with LJ041, and arrow indicates the biofilm on PET (Panel 2F) SEM image revealed that KT2440 does not form biofilm on PET (Panel 2G) SEM observation of LJ041 biofilm forming cells on PET (Panel 2H) SEM observation of fragmenting PET film (highlighted area with arrow) by LJ041 (Panel 2I) LJ041 forms holes on PET film (Panel 2J) HPLC chromatographs of PET-degraded products after 24 h and 72 h. Experiments were conducted in 5 mL M9 medium containing 20 mM glucose and about 60 mg of amorphous PET particle. 
         FIG. 3  depicts strain LJ041 that was tested for selective degradation of BHET to TPA. The LJ041 strain converted BHET to TPA at 3-fold higher rate relative to wild-type  P. putida  KT2440 (LJ041:12.8 mg/L/h vs KT2440: 4.7 mg/L/h). 
         FIG. 4  depicts Engineered TPA catabolic pathway in  P. putida  KT2440, transporter TpaK and catabolic genes (TphA1, TphA2, TphA3, and TphB) are originally from  R. jostii  RHA1 and  Comamonas  sp. strain E6, respectively. 
         FIG. 5  depicts Engineered  P. putida  KT2440 strain enables TPA utilization. (A) Growth curves of the strain (B) growth rate of the strains (C) TPA utilization of the strains. Growth of the strains was assessed in minimal medium containing either 10 mM TPA or 10 mM PCA as the sole substrate for growth, and TPA utilization was measured during growth in minimal medium with 10 mM TPA as the sole growth substrate. Concentrations of TPA were measured using high performance liquid chromatography (HPLC) by injecting culture supernatant onto a Rezex RFQ-Fast Acid H+ (8%) HPLC column. Mobile phase consisted of 5 mM H 2 SO 4 , and samples were run at 0.6 ml/min at 60° C. TPA eluted at ˜21 minutes and was detected at a wavelength of 230 nm via a UV-Vis detector. Area under the elution peak was integrated and TPA concentration was calculated against a standard. 
         FIG. 6A  depicts codon optimized sequences of PETase (SEQ ID NO: 1) and MHETase (SEQ ID NO: 2) genes from  Ideonella sakaiensis  201-F6 to  P. putida  KT2440. 
         FIG. 7  depicts a plasmid map of pLJ080. 
         FIG. 8  depicts the nucleotide sequence of plasmid pLJ080 (SEQ ID NO: 3). 
         FIG. 9  depicts the amino acid sequences of PETase (SEQ ID NO: 4) and MHETase (SEQ ID NO: 5). 
         FIG. 10  depicts a plasmid map of pLJ081. 
         FIG. 11  depicts the plasmid sequence (SEQ ID NO: 6) of PETase with GFP tag (pLJ081). 
         FIG. 12  depicts (SEQ ID NO: 7) the nucleotide sequence of synthetic tphC II  gene. 
         FIG. 13  depicts (SEQ ID NO: 8) the nucleotide sequence of synthetic tphA2 II  gene. 
         FIG. 14  depicts (SEQ ID NO: 9) the nucleotide sequence of synthetic tphA3 II  gene. 
         FIG. 15  depicts (SEQ ID NO: 10) the nucleotide sequence of synthetic tphB II  gene. 
         FIG. 16  depicts (SEQ ID NO: 11) the nucleotide sequence of synthetic tphA1 II  gene. 
         FIG. 17  depicts (SEQ ID NO: 12) the nucleotide sequence of synthetic tpiB gene. 
         FIG. 18  depicts (SEQ ID NO: 13) the nucleotide sequence of synthetic tpiA gene. 
         FIG. 19  depicts (SEQ ID NO: 14) the nucleotide sequence of the local chromosomal sequence in strain IP103. Homology arms sequences are shown in italic. Synthetic ribosome binding sites are shown in bold. Coding sequences for tph genes are underlined. 
         FIG. 20  depicts (SEQ ID NO: 15) the nucleotide sequence of the local chromosomal sequence in strain IP131. Homology arms sequences are shown in italic. Synthetic ribosome binding sites are shown in bold. Coding sequences for tph, tpi and kanamycin selection marker genes are underlined. 
         FIG. 21  depicts growth and TPA concentration in a medium containing an engineered Acinetobacter baylyi ADP1 strain, IP103, expressing the tphC II A2 II A3 II B II A II  synthetic genes was grown in  Acinetobacter  minimal media in the presence of 5 mM terephthalic acid and 20 mM pyruvate. 
         FIG. 22  depicts TPA consumption over time of an engineered  Acinetobacter baylyi  ADP1 strain, IP 131, expressing the synthetic terephthalate transporter genes, tpiAB, as well as the tphC II A2 II A3 II B II A II  genes, and the parent strain, IP103, expressing only the tphC II A2 II A3 II B II A II  genes, were grown in Acinetobacter minimal media supplemented with 5 mM terephthalic acid and 20 mM pyruvate. The strains were fed only at the beginning of the experiment. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that some embodiments as disclosed herein may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the embodiments described herein should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein. 
     In an embodiment, disclosed herein is an engineered  P. putida  KT2440 co-expressing PETase and MHETase enzymes that selectively degrades PET into monomers, ethylene glycol and terephthalate (TPA). In another embodiment, disclosed herein are methods for making and using a highly efficient EG metabolizing  P. putida  KT2440 strain. Given that native  P. putida  does not have a TPA metabolic pathway, nor the proteins to transport TPA into the cell, the next metabolic engineering challenge for developing synthetic  P. putida  strain to plastic upcycling was enabling TPA catabolism in  P. putida  KT2440. TPA transporters and catabolic pathway have been characterized in several microorganisms including  Comamonas  sp. strain E6 and  Rhodococcus jostii  RHA1. 
     In an embodiment, disclosed herein are engineered  P. putida  KT2440 strains that use TPA through heterologous expression of a TPA transporter from  Rhodococcus jostii  RHA1 and catabolic genes from  Comamonas  sp. E6 ( FIG. 4 ). In an embodiment, the pcaIJ gene was knocked out in the engineered strains, enabling the biological conversion of TPA to β-ketoadipate. Ultimately, the engineered strains disclosed herein enable the upcycling of PET-derived TPA into atom-efficient β-ketoadipic acid, a high-value chemical that can be used to produce a biodegradable plastic material with superior properties. 
     Disclosed herein, in an embodiment, TPA catabolism is enabled in  P. putida  KT2440 by heterologous expression of TPA transporters (tpaK) and catabolic genes cluster I or II from  R. jostii  RHAI and  Comamonas  sp. E6, respectively. The engineered, non-naturally occurring strains can use TPA as a sole carbon source and use TPA at about 0.05 g L −1  h −1 . In an embodiment, the pcaIJ gene was knocked out in an engineered TPA utilizing strain. The strain could convert TPA to β-ketoadipate. In another embodiment, TPA utilization strain can be engineered for consolidated bioprocessing of PET by enabling selective degradation of PET and ethylene glycol utilization. In an embodiment, strains could be evolved to enhance TPA catabolic rates. 
     The present disclosure also relates to a biological strategy for degrading PET, which can subsequently enable atom-efficient biological transformations to novel intermediates (e.g., β-ketoadipate and/or muconate), which may be converted to high strength composites. PETase hydrolyses PET to produce bis(2-hydroxyethyl) terephthalate (BHET), mono-(2-hydroxyethyl) terephthalate (MHET), terephthalate (TPA), and ethylene glycol (EG), and MHETase catalyzes MHET to TPA and EG. Hence, as shown herein, co-expression of PETase and MHETase in an engineered strain can enable PET degradation to TPA and EG. Thus, in some embodiments of the present disclosure, a biological method is provided for the selective degradation of PET into PET monomers via co-expression and secretion of PETase and MHETase in  Pseudomonas putida,  which can grow well in simple minimal salt medium. 
     Therefore, the present disclosure relates to biological methods for the selective degradation of PET into PET monomers via co-expression PETase and MHETase in  Pseudomonas putida,  which can grow well in simple minimal salt medium. Among other things,  I. sakaiensis  PETase, ISF6_4831 and MHETase, ISF6_0224 genes were codon optimized for expression in KT2440 including their secretion signal peptides, which are compatible to the  P. putida  chaperone SecB-dependent secretion system. In addition, the genes were integrated into the  P. putida  genome with the tac promoter to enable constitutive expression. In certain embodiment, the term “tac”, “Ptac” and “P-Tac” may be used interchangeable to mean a tac promoter. The developed LJ041 strain formed a biofilm on PET. LJ041 enables highly-selectively degradation of PET into monomer TPA via BHET and MHET and confirmed secretion of PETase and MHETase enzymes via the chaperone-dependent native  P. putida  secreting system. These innovations could lead to a  P. putida  strain for selective biological degradation and conversion of PET into bio-derived chemical building blocks. 
       I. sakaiensis  PETase, ISF6_4831 and MHETase, ISF6_0224 genes were codon optimized to KT2440 including their secretion signal peptides, which are compatible to the  P. putida  chaperone Sec-dependent secretion system. To confirm secretion of codon optimized PETase in  P. putida  via the  I. sakaienesis  secretion signal peptide, green fluorescent protein (GFP) was genetically linked to the C-terminus of PETase and expressed in  P. putida.  Efficient secretion of GFP-tagged PETase was confirmed via microscopy and immunoprecipitation, see  FIG. 1 : Panel A illustrates bright field microscopic observation of the strain expressing PETase with GFP tag; Panel B illustrates microscopic observation of GFP signal of the strain expressing PETase with GFP tag; Panel C illustrates GFP signal of the supernatant of wild-type strain and the strain expressing GFP tagged PETase; Panel D illustrates immunoprecipitation of GFP tagged PETase with GFP specific GFP-Trap® (ChromoTek GmbH, Planegg-Martinsried, Germany); and Panel E illustrates a microscopic image of PET particle incubated with the strain expressing GFP tagged PETase. 
     Next, referring to  FIG. 2 , the codon optimized PETase and MHETase genes were successfully integrated into the  P. putida  genome with the tac promoter to enable constitutive expression, and obtained the LJ041 strain (see Panel A). LJ041 formed a biofilm (see  FIG. 2 , Panels B, E, and G) on amorphous PET coupon and visually observed the fragmenting PET (see  FIG. 2 , Panels C and H). HPLC analysis revealed that LJ041 enabled highly-selectively degradation of PET into monomer TPA via BHET and MHET (see  FIG. 2 , Panel J). These results indicate that the codon-optimized signal sequences (which are codon optimized to KT2440), “ATGAACTTCCCTCGCGCGTCGCGCCTGATGCAGGCGGCGGTCCTCGGTGGTCTGAT GGCAGTCAGCGCCGCGGCCACC”, which encode “MNFPRASRLMQAAVLGGLMAVSAAATA”, and “ATGCAGACCACCGTCACCACTATGCTGCTGGCATCGGTCGCCCTGGCCGCC”, which is enclosed signal peptide “MQTTVTTMLLASVALAA”, for MHETase, respectively, are sufficient for enzyme secretion. These secretion signal peptides may be used for trafficking other proteins in  P. putida  via the Sec-dependent native  P. putida  secreting system. Of note,  Ideonella sakaiensis  201-F6 grows only in rich-medium but not in the minimal salt medium (data not shown). Thus, the LJ014 has an advantage over the  Ideonella sakaiensis  201-F6 as an industrial biocatalyst to degrade PET and to subsequently upgrade the degradation products into high-value chemicals. In addition, we introduced PETase and MHETase encoding genes into the genome of  P. putida  EM42 strain via deploying pLJ080 plasmid, the genome reduced version of  P. putida  KT2440, and developed LJ042 strain. 
       FIG. 2  illustrates degradation results of PET by LJ041 (Panel A) integrated gene cassette (Panel B) visual observation of biofilm of 1141 on PET film (arrow) (Panel C) fragmenting PET by LJ041 (Panel D) SEM observation of PET particles cultured with KT2440, after 5 days of incubation (Panel E) SEM observation of PET cultured with LJ041, and arrow indicates the biofilm on PET (Panel F) SEM image revealed that KT2440 does not form biofilm on PET (Panel G) SEM observation of LJ041 biofilm forming cells on PET (Panel H) SEM observation of fragmenting PET film (highlighted area with arrow) by LJ041 (Panel I) LJ041 forms holes on PET film (Panel J) HPLC chromatographs of PET-degraded products after 24 h and 72 h. Experiments were conducted in 5 mL M9 medium containing 20 mM glucose and about 60 mg of amorphous PET particle. 
     Next, the LJ041 strain was tested for selective degradation of BHET to TPA (see  FIG. 3 ). The LJ041 strain converted BHET to TPA at 3-fold higher rate relative to wild-type  P. putida  KT2440 (LJ041:12.8 mg/L/h vs KT2440: 4.7 mg/L/h). Taken together, this innovation could lead to a  P. putida  strain for selective biological degradation and conversion of PET into bio-derived chemical building blocks. 
     Materials and Methods: 
     Plasmid construction: Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers synthesized by Integrated DNA Technologies (IDT) were used in all PCR amplification. Plasmids were constructed using Gibson Assembly® Master Mix (New England Biolabs) according to the manufacturer&#39;s instructions. Primers used for PCR amplification and Gibson assembly are listed in Table 1. The vector, pBLT-2 (Addgene plasmid # 22806) was used for plasmid-based overexpression of PETase with a green fluorescence protein (GFP) tag. Plasmids for gene integration were constructed in pK18sB, which is unable to replicate in  P. putida  KT2440, and contains the kanamycin-resistant marker to select for integration of the plasmid into the genome by homologous recombination and sacB to counter select for a second recombination event to subsequently remove the plasmid backbone from the genome. Detail of plasmids construction is provided in Table 2. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of Primers 
               
            
           
           
               
               
            
               
                 Primer ID 
                 5′-3′ 
               
               
                   
               
               
                 oLJ227 
                 GACATGATTACGAATTCGAGCTCGGTACCCGTGCGATTA 
               
               
                   
                 CTGTGGGAG 
               
               
                   
               
               
                 oLJ232 
                 CCGGAGGCTTTTGACTCGGAGGCGCGGCGCAGGC 
               
               
                   
               
               
                 oLJ228 
                 CGGATAACAATTTCACACTGAGTATTGCCTGAACCG 
               
               
                   
               
               
                 oLJ229 
                 TTCAGGCAATACTCAGTGTGAAATTGTTATCCGCTCACA 
               
               
                   
                 ATTCCACACATTATACGAGCCGATGATTAATTGTCAACA 
               
               
                   
                 GCTCTTCATCAAGTCAAAACACTATATAGGAACG 
               
               
                   
               
               
                 oLJ230 
                 ATGTAATCCTTGTTATAGGCTGCAGTTCGCAGTGCG 
               
               
                   
               
               
                 oLJ231 
                 ACTGCGAACTGCAGCCTATAACAAGGATTACATATAAGG 
               
               
                   
                 GTATATCAAATGCAGACCACCGTCACC 
               
               
                   
               
               
                 oLJ233 
                 TGCGCCGCGCCTCCGAGTCAAAAGCCTCCGGTCGGAGGC 
               
               
                   
                 TTTTGACTTCAAAACCACCCTGCTGTCGATG 
               
               
                   
               
               
                 oLJ234 
                 CGGCCAGTGCCAAGCTTGCATGCCTGCAGGAAATCTAAC 
               
               
                   
                 TGCCTTCGCCC 
               
               
                   
               
               
                 oLJ406 
                 TATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACAC 
               
               
                   
                 TTTCATCAAGTCAAAACACTATATAGGAACGAAAC 
               
               
                   
               
               
                 oLJ407 
                 TCCGCACTGCGAACTGCAGCGGTGGTTCTGAGGAATCTT 
               
               
                   
                 ACATGAGC 
               
               
                   
               
               
                 oLJ408 
                 GTAAGATTCCTCAGAACCACCGCTGCAGTTCGCAGTGCG 
               
               
                   
               
               
                 oLJ409 
                 AGTCCAGTTACGCTGGAGTCTGAGGCTCGTCCTGAATGA 
               
               
                   
                 TCTACTTGTAGAGTTCGTC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Plasmid construction details 
               
            
           
           
               
               
               
            
               
                 Plasmid 
                 Purpose 
                 Construction detail 
               
               
                   
               
               
                 pLJ080 
                 Genome 
                 The PETase genes cassette was amplified with 
               
               
                   
                 integration 
                 primers oLJ229 (Fwd) and oLJ230 (Rev), and 
               
               
                   
                 of over- 
                 MHETase oLJ231 (Fwd) and oLJ232 (Rev) using 
               
               
                   
                 expressing 
                 synthesizes gBlock as a temple. The 5′ homology 
               
               
                   
                 cassette of 
                 region was amplified from  P. putida  KT2440 
               
               
                   
                 PETase 
                 genomic DNA with primers oLJ227(Fwd), and 
               
               
                   
                 and 
                 oLJ228 (Rev), and 3′ homology region was 
               
               
                   
                 MHETase 
                 amplified with oLJ233 (Fwd) and oLJ234 (Rev). 
               
               
                   
                   
                 These products were assembled into pK18sB 
               
               
                   
                   
                 digested with SmaI and SalI. 
               
               
                 pLJ081 
                 Over- 
                 A DNA fragment containing the PETase genewas 
               
               
                   
                 expressing 
                 amplified from pLJ080 with primers oLJ406 
               
               
                   
                 PETase-GFP 
                 (Fwd) and oLJ407 (Rev), and GFP gene fragment 
               
               
                   
                   
                 was obtained with primers oLJ408 (Fwd) and 
               
               
                   
                   
                 oLJ409 (Rev), amplified from GFP containing 
               
               
                   
                   
                 plasmid. This product was assembled into 
               
               
                   
                   
                 pBLT-2 digested with XbaI and EcoRV. 
               
               
                   
               
            
           
         
       
     
     The PETase and MHETase genes from  Ideonella sakaiensis  201-F6 were codon optimized to  P. putida  KT2440 using online program Optimizer with a random approach (http://genomes.urv.es/OPTIMIZER/), gene fragments were synthesized at Integrated DNA Technologies, Inc, and obtained the double-stranded and linear gBlock, see  FIG. 6 . The plasmid used for of integration of codon optimize PETase and MHETase to  P. putida  KT2440 contain the approximately 0.7 kb homology region on either side of the intergenic region immediately after PP 1642 and PP 1643 of  P. putida  KT2440. Features include the tac promoter to drive gene expression and a tonB terminator situated behind the fragments cloned into the plasmid backbone, which are depicted in  FIG. 7 . Synthetic ribosomal binding site (sRBS) were designed using an online program from the Salis laboratory at Penn State University, in front of genes, the designed sRBS (TCATCAAGTCAAAACACTATATAGGAACGAAACC) of PETase was predicted to have a translation initiation rate (TIR) of 27306.09, and MHETase has a sRBS (TAACAAGGATTACATATAAGGGTATATCAA) with TIR of 32480.74. Plasmid sequence of pLJ80 is provided in Table S5 in the Appendix. The protein sequences of PETase and MHETase are provided in  FIG. 8 . Plasmid was transformed into competent NEB 5-alpha F′I q    E. coli  (New England Biolabs) according to the manufacturer&#39;s instructions. Transformants were selected on LB plates containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 15 g/L agar, supplemented with 50 μg/mL kanamycin grown at 37° C. The sequences of all plasmid inserts were confirmed using Sanger sequencing (GENEWIZ, Inc.). 
     Strain construction:  P. putida  KT2440 (ATCC 47054) was used as the basis of strain engineering and gene replacements were made using the antibiotic/sacB system of selection and counter-selection. In an embodiment, the properties and description of some strains disclosed herein is depicted in Table 3. To prepare electrocompetent cells of  P. putida  KT2440 strains, a modified sucrose-based protocol was used. The plasmid was introduced to competent cells via electroporated at 1.6 kV, 25 μF, 200 Ohms. The transformation was plated on an LB agar plate containing 50 μg/ml kanamycin antibiotics and incubated at 30° C. overnight. Initial colonies from the transformation plates were re-streaked on selective LB agar plates and grown at 30° C. overnight to obtain clonal transformants. For sucrose counter-selection, clonal transformants were streaked on YT plates containing 25% (YT+25%; w/v) sucrose (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, 18 g/L agar), and incubated at 30° C. overnight. The single colony of  P. putida  KT2440 containing the PETase and MHETase genes were successfully isolated. The strain was analyzed for the correct gene replacement by performing a colony PCR at the site of integration. The LJ102 was constructed by transforming pLJ081 plasmid into  P. putida  KT2440, the plasmid map and sequence are provided in  FIG. 10  and  FIG. 11 . 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Strains 
               
            
           
           
               
               
               
            
               
                 Strain 
                   
                   
               
               
                 ID 
                 Genotype 
                 Description of strain 
               
               
                   
               
               
                 KT2440 
                   P. putida  KT2440 
                 Wild-type  P. putida  KT2440 
               
               
                   
                   
                 (ATCC 47054) 
               
               
                 EM42 
                   P. putida  KT2440 
                 Genome reduced strain derived from 
               
               
                   
                 Δprophage1-4 
                   P. putida  KT2440 obtained from 
               
               
                   
                 Δflagellum 
                 Victor de Lorenzo&#39;s laboratory (Centro 
               
               
                   
                 ΔendA-1 
                 Nacional de Biotecnología 
               
               
                   
                 ΔendA-2 ΔTn7 
                 (CNB-CSIC), Madrid, Spain) 
               
               
                   
                 ΔhsdRMS ΔTn4652 
               
               
                 LJ102 
                 KT2440 + pBTL-2- 
                 KT2440 containing the pBTL-2 plasmid 
               
               
                   
                 PETase_GFP 
                 with PETase and GFP 
               
               
                 LJ041 
                 KT2440 1642::Ptac:: 
                 KT2440 with the PETase and MHETase 
               
               
                   
                 PETase-MHET 
                 cassette integrated within the intergenic 
               
               
                   
                   
                 region between PP_1642 and PP_1643 
               
               
                 LJ042 
                 EM42 PP 1642::Ptac:: 
                 EM42 with the PETase and MHETase 
               
               
                   
                 PETase-MHET 
                 cassette integrated within the intergenic 
               
               
                   
                   
                 region between PP_1642 and PP_1643 
               
               
                   
               
            
           
         
       
     
     PET and BHET degradation experiment: To assess the selective degradation of PET/BHET by the PETase and MHETase expressing strain, shake flask experiments were performed using 125 mL baffled flasks containing 25 mL modified M9 media (6.78 g/L Na 2 HPO 4 , 3.00 g/L K 2 HPO 4 , 0.50 g/L NaCl, 1.66 g/L NH 4 Cl, 0.24 g/L MgSO 4 , 0.01 g/L CaCl 2 , and 0.002 g/L FeSO 4 ) supplemented with 20 mM of glucose and amorphous PET coupons (amorphous PET films with a crystallinity of 14.8±0.2%, synthesized at NREL) or BHET (Obtained from IBM Almaden Research Center, BHET was derived from waste PET bottles via chemical depolymerization process), and inoculated to OD 600  0.1 with pre-culture. Pre-cultures of the strains were prepared by inoculating 25 mL M9 medium supplemented with 20 mM glucose in a 125 mL baffled flask to an OD 600  of 0.05-0.1 and incubating shaking at 225 rpm, 30° C. At mid log phase (OD 600  0.5-1.0) cells were harvested by centrifugation at 13,000 rpm, and the cell pellets were washed twice and resuspended in M9 medium without a carbon source. Cultures were incubated shaking at 225 rpm, 30° C. 1 mL samples were collected periodically and subjected to HPLC analysis to detect the degraded products. After the fermentation, PET coupons were subjected to microscopic observation. 
     Scanning Electron Microscopy (SEM): Imaging by scanning electron microscopy (SEM) was performed using a FEI Quanta 400 FEG instrument under low vacuum (0.45 Torr) operating with the gaseous solid-state detector (GAD). Samples were prepared for imaging by fixation in 2.5% gluteraldehyde buffered in 1× PBS (EMS, Hatfield, PS), dehydration in an ethanol series, then freezing in liquid nitrogen followed by lyophilization. Dry samples were mounted on aluminum stubs using carbon tape, and sputter coated with 9 nm of Ir metal. Images were captured at a beam accelerating voltage of 24 keV. 
     High performance liquid chromatography (HPLC) analysis: Concentrations of TPA, MHET, and BHET were measured using HPLC by injecting 6 μL of 0.2-μm filter-sterilized culture supernatant onto an Agilent1100 series system (Agilent USA, Santa Clara, Calif.) equipped with a Phenomenex Rezex RFQ-Fast Fruit H+ column (Phenomenex, Torrance, Calf.) and cation H+ guard cartridge (Bio-Rad Laboratories, Hercules, Calif.) at 85° C. A mobile phase of 0.1N sulfuric acid was used at a flow rate of 1.0 mL/min. Diode array detectors were used for compound detection. Compounds were identified by relating the retention times and spectral profiles with standard HPLC grade pure compounds (Sigma Aldrich, St. Louis, Mo., USA) and the concentration of each compound was calculated based on a calibration curves generated using pure compounds. 
     To enable TPA catabolism in  P. putida  KT2440, genes for TPA transport and for conversion of TPA into protocatechuic acid (PCA), an intermediate metabolite of β-ketoadipate pathway were introduced into the chromosome of  P. putida  strain KT2440. Three different operons containing genes required for TPA catabolism [two operons from  Comamonas  sp. E6 (operon I: tphA2I, tphA3I, tphBI, and tphA1I) and (operon II: tphA2II, tphA3II, tphBII, and tphA1II), and one from  R. jostii  RHA1 (tpaA1, tpaA2, tpaC, and tpaB)], and two different operons containing transport genes [one from  Comamonas  sp. E6 (tphC, tpiA, and tpiB) and one from  R. jostii  RHA1(tpaK) were tested in various combinations (Table 4). Additionally, each operon was placed under control of 3 different promoters of varying strengths (from strongest to weakest: P-Tac, P-549, P-Lac, P-3079). Those gene clusters were successfully integrated into a modified version of  P. putida  KT2440 that has 3 poly-attB genetic islands for DNA insertion via highly efficient phage integrase system. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Generated strains of  P. putida  containing genes for terephthalic acid transport 
               
               
                 and catabolism under control of promoters with varying strengths. 
               
            
           
           
               
               
               
               
            
               
                   
                 Catabolic Genes 
                 Transport Gene(s) 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Source 
                   
                   
                 Source 
                   
                   
                 TPA 
               
               
                 TDM# 
                 Organism 
                 Operon 
                 Promoter 
                 Organism 
                 Operon 
                 Promoter 
                 growth 
               
               
                   
               
               
                 56 
                 
                   Comamonas 
                 
                 tphA2 I A3 I B I A1 I   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-549 
                 No 
               
               
                 57 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 58 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 59 
                 
                   Comamonas 
                 
                 tphA2 II A3 II B II A1 II   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-549 
                 No 
               
               
                 60 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 61 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 62 
                 
                   Rhodococcus 
                 
                 tpaA1A2CB 
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-549 
                 No 
               
               
                 63 
                   jostii  RHA1 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 64 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 65 
                 
                   Comamonas 
                 
                 tphA2 I A3 I B I A1 I   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-Lac 
                 No 
               
               
                 66 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 67 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 68 
                 
                   Comamonas 
                 
                 tphA2 II A3 II B II A1 II   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-Lac 
                 No 
               
               
                 69 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 70 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 71 
                 
                   Rhodococcus 
                 
                 tpaA1A2CB 
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-Lac 
                 No 
               
               
                 72 
                   jostii  RHA1 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 73 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 74 
                 
                   Comamonas 
                 
                 tphA2 I A3 I B I A1 I   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-3079 
                 No 
               
               
                 75 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 76 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 77 
                 
                   Comamonas 
                 
                 tphA2 II A3 II B II A1 II   
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-3079 
                 No 
               
               
                 78 
                 sp. E6 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 79 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 80 
                 
                   Rhodococcus 
                 
                 tpaA1A2CB 
                 P-Tac 
                 
                   Comamonas 
                 
                 tphC- 
                 P-3079 
                 No 
               
               
                 81 
                   jostii  RHA1 
                   
                 P-Tac 
                 sp. E6 
                 tpiBA 
                   
                 No 
               
               
                 82 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 83 
                 
                   Comamonas 
                 
                 tphA2 I A3 I B I A1 I   
                 P-Tac 
                 
                   Rhodococcus 
                 
                 tpaK 
                 P-549 
                 Yes 
               
               
                 84 
                 sp. E6 
                   
                 P-Tac 
                   jostii  RHA1 
                   
                   
                 Yes 
               
               
                 85 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 86 
                 
                   Comamonas 
                 
                 tphA2 II A3 II B II A1 II   
                 P-Tac 
                 
                   Rhodococcus 
                 
                 tpaK 
                 P-549 
                 Yes 
               
               
                 87 
                 sp. E6 
                   
                 P-Tac 
                   jostii  RHA1 
                   
                   
                 Yes 
               
               
                 88 
                   
                   
                 P-Lac 
                   
                   
                   
                 No 
               
               
                 89 
                 
                   Rhodococcus 
                 
                 tpaA1A2CB 
                 P-Tac 
                 
                   Rhodococcus 
                 
                 tpaK 
                 P-549 
                 No 
               
               
                 90 
                   jostii  RHA1 
                   
                 P-Tac 
                   jostii  RHA1 
                   
                   
                 No 
               
               
                   
               
            
           
         
       
     
     In an embodiment, thirty-five strains were generated, of which four had substantial growth with TPA as the sole carbon source. Each of the four strains that were able to metabolize TPA contained one of the two  Comamonas  sp. E6 catabolic operons (I or II) in combination with the  R. jostii  transporter. Robust expression was a requirement for TPA utilization, as growth was only detected when catabolic and transport genes were expressed from the strongest tested promoters (P-Tac or P-549). Of note, the growth data revealed that neither  Comamonas  sp. E6 TPA transporter nor  R. jostii  RHAI catabolic genes enable TPA catabolism in  P. putida  KT2440. Growth in minimal media containing either 10 mM TPA or 10 mM PCA was compared for each of the TPA catabolizing strains. An extended lag phase and about a 3-fold slower growth rate for all strains indicated that TPA is not used as efficiently as PCA as a substrate ( FIGS. 5A and 5B , Table 5). However, quantification of TPA from late exponential phase cultures grown in minimal media with 10 mM TPA indicated that about 90% of TPA was consumed ( FIG. 5C ). Ongoing experiments are aimed at optimizing import and processing of TPA. Additionally, the ultimate objective of this project is to use  P. putida  for the valorization of TPA into other high value products, such as β-ketoadipate. To that end, the genes that facilitate β-ketoadipate consumption, pcaIJ, have been deleted from the TPA utilizing strains to allow ↑-ketoadipate accumulation, and the strains have been confirmed by PCR. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Growth characteristics of TPA utilizing strains of 
               
               
                   P. putida  in minimal medium containing either 10 
               
               
                 mM TPA or 10 mM PCA as the sole growth substrate. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Lag 
                 Growth 
                 Doubling 
               
               
                 Strain 
                 Substrate 
                 Phase (h) 
                 Rate (h −1 ) 
                 Time (h) 
               
               
                   
               
               
                 TDM083 
                 TPA 
                 16.4 ± 0.1 
                 0.108 ± 0.002 
                 6.41 ± 0.13 
               
               
                 TDM084 
                 TPA 
                 16.4 ± 0.8 
                 0.102 ± 0.003 
                 6.81 ± 0.20 
               
               
                 TDM086 
                 TPA 
                 17.4 ± 0.9 
                 0.099 ± 0.003 
                 7.01 ± 0.19 
               
               
                 TDM087 
                 TPA 
                 17.6 ± 0.5 
                 0.099 ± 0.001 
                 6.98 ± 0.07 
               
               
                 KT2440 
                 TPA 
                 No Growth 
                 No Growth 
                 No Growth 
               
               
                 TDM083 
                 PCA 
                 2.8 ± 0.0 
                 0.395 ± 0.024 
                 1.76 ± 0.10 
               
               
                 TDM084 
                 PCA 
                 2.8 ± 0.0 
                 0.378 ± 0.026 
                 1.84 ± 0.13 
               
               
                 TDM086 
                 PCA 
                 2.9 ± 0.1 
                 0.327 ± 0.066 
                 2.17 ± 0.40 
               
               
                 TDM087 
                 PCA 
                 2.8 ± 0.3 
                 0.311 ± 0.029 
                 2.24 ± 0.22 
               
               
                 KT2440 
                 PCA 
                 2.6 ± 0.3 
                 0.300 ± 0.010 
                 2.31 ± 0.08 
               
               
                   
               
            
           
         
       
     
     Different versions of a synthetic operon coding for a terephthalic acid degradation pathway were constructed for chromosomal integration and expression in  Acinetobacter baylyi  ADP1. This operon includes codon-optimized versions of the genes tphC II A2 II A3 II B II A II  and tpiBA from  Comamonas  sp. E6 under control of a constitutive promoter, with each gene being preceded by a synthetic ribosome binding site sequence. The description and accession numbers for the wild-type  Comamonas  sp. E6 tphC II A2 II A3 II B II A II  and tpiBA genes are listed in Table 6. For the homologous recombination and insertion of the operon in the chromosome of  Acinetobacter baylyi  ADP1, upstream and downstream homology arms of ˜2000 bp were amplified from genomic DNA and assembled by overlap extension PCR to flank the synthetic genes. Linear DNA fragments were transformed into naturally competent  Acinetobacter baylyi  ADP1 cells as described in the literature. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Protein 
                   
               
               
                   
                 accession 
               
               
                 Gene 
                 number 
                 Description 
               
               
                   
               
             
            
               
                 tphC II   
                 BAE47084.1 
                 Periplasmic terephthalate binding receptor 
               
               
                 tphA2 II   
                 BAE47085.1 
                 Oxygenase large subunit of terephthalate 
               
               
                   
                   
                 1,2-dioxygenase 
               
               
                 tphA3 II   
                 BAE47086.1 
                 Oxygenase small subunit of terephthalate 
               
               
                   
                   
                 1,2-dioxygenase 
               
               
                 tphB II   
                 BAE47087.1 
                 1,2-dihydroxy-3,5-cyclohexadiene-1,4- 
               
               
                   
                   
                 dicarboxylate dehydrogenase 
               
               
                 tphA1 II   
                 BAE47088.1 
                 Reductase component of terephthalate 
               
               
                   
                   
                 1,2-dioxygenase 
               
               
                 tpiB 
                 BAN66715.1 
                 Small transmembrane protein of the aromatic 
               
               
                   
                   
                 acids transporter 
               
               
                 tpiA 
                 BAN66716.1 
                 Large transmembrane protein of the aromatic 
               
               
                   
                   
                 acids transporter 
               
               
                   
               
            
           
         
       
     
     In a first shake-flask experiment, an engineered  Acinetobacter baylyi  ADP1 strain, IP103, expressing the tphC II A2 II A3 II B II A II  synthetic genes was grown in  Acinetobacter  minimal media in the presence of 5 mM terephthalic acid and 20 mM pyruvate, the latter being fed every 24 hours to support cell growth. As seen in  FIG. 1 , more terephthalic acid was consumed by IP103 than by the wild-type strain. The slight decrease in TPA concentration for the wild-type strain is an effect of the dilution caused by feeding daily with 20 mM pyruvate to support cell growth. 
     Genes expressing the terephthalate transporter from  Comamonas  sp. E6, tpiBA, were then similarly codon optimized and incorporated into the genome of IP103 downstream of the tphC II A2 II A3 II B II A II  genes, such that expression of all of these genes was driven as an operon by the same promoter. In a shake-flask experiment, this new strain expressing the synthetic terephthalate transporter genes, tpiAB, as well as the tphC II A2 II A3 II B II A II  genes, IP131, and the parent strain expressing only the tphC II A2 II A3 II B II A II  genes, IP103, were grown in  Acinetobacter  minimal media supplemented with 5 mM terephthalic acid and 20 mM pyruvate, fed only at the beginning of the experiment. As seen in  FIG. 2 , IP131 was able to degrade terephthalic acid more quickly, than IP103, indicating that expression of the terephthalate transporter improved the ability of this strain to metabolize this substrate. 
     The foregoing discussion and examples have been presented for purposes of illustration and description. The foregoing is not intended to limit the aspects, embodiments, or configurations to the form or forms disclosed herein. In the foregoing Detailed Description for example, various features of the aspects, embodiments, or configurations are grouped together in one or more embodiments, configurations, or aspects for the purpose of streamlining the disclosure. The features of the aspects, embodiments, or configurations, may be combined in alternate aspects, embodiments, or configurations other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention that the aspects, embodiments, or configurations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment, configuration, or aspect. While certain aspects of conventional technology have been discussed to facilitate disclosure of some embodiments of the present invention, the Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect, embodiment, or configuration.