IN THE UNITED STATES PATENT & TRADEMARK OFFICE

PCT INTERNATIONAL PATENT APPLICATION

METHODS FOR ENZYMATIC AND MICROBIAL DEGRADATION OF

POLYETHYLENE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/989,172 filed on March 13, 2020, which is hereby incorporated by reference in its entirety.

FIELD

[0002] This invention relates to compositions and processes for degrading plastic products comprising polyethylene. The invention further relates to the enzymatic and/or microbial degradation of polyethylene that is the most common plastic in use.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY [0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing filename: ZYMR_063_01WO_SeqList_ST25.txt, date recorded, March 11, 2021, file size ~ 154 kilobytes.

BACKGROUND OF THE DISCLOSURE

[0004] Polyethylene (PE) makes up over one-third of the total plastics market globally and is one of the most durable plastic materials available. However, this market share and expansive utility make PE one of the most prevalent plastics polluting the environment. The methods by which the global economy disposes of PE are rudimentary, at best. Typically, PE industrial waste is burned, which only compounds the negative environmental impacts of this ubiquitous material. Consequently, there is a critical global need for environmentally sustainable solutions for PE degradation.

BRIEF SUMMARY OF THE DISCLOSURE

[0005] The disclosure provides an answer to this pressing global environmental problem. By using biology to degrade PE, the disclosure teaches an environmentally sound solution to this pressing crisis. Thus, in aspects, the disclosure provides for microbes that are able to degrade PE. In other aspects, the disclosure provides for various enzymes that are capable of degrading PE. The disclosure also contemplates various synergisms between utilizing both a PE degrading microbe and a PE degrading enzyme in conjunction. As will be seen in the taught methods, compositions, and microbes, the disclosure provides an elegant solution to a vexing worldwide environmental problem.

[0006] In aspects, the disclosure comprises methods for the degradation of polyethylene (PE) using enzymes and/or microbe strains. For example, in aspects of the disclosure, the following embodiments are taught: (1) Use of any Pseudomonas nitroreducens species to degrade PE, (2) Use of a specific strain of Pseudomonas citronellolis (ATCC 13674 = NRRL B-2504 = DSM 50332) to degrade PE, (3) Use of any P. nitroreducens or P. citronellolis species to degrade a PE powder, (4) Use of any Pseudomonas species to degrade PE by reducing its molecular weight, (5) Growth conditions used for P. nitroreducens and P. citronellolis to facilitate growth on and/or degradation of PE, (6) Use of a laccase enzyme to degrade PE powder, (7) Use of a laccase enzyme along with/ ⁵ . citronellolis or P. nitroreducens to degrade PE, (8) Use of UV to degrade PE powder, and (9) Use of UV along withi ⁵ . citronellolis or P. nitroreducens to degrade PE.

[0007] The present disclosure provides a method of degrading polyethylene, comprising: (a) combining a Pseudomonas microbe and/or an oxidase enzyme with a polyethylene substrate. In some embodiments, the polyethylene substrate’s molecular weight is reduced upon exposure to the, Pseudomonas microbe and/or the oxidase enzyme.

[0008] In some embodiments, the Pseudomonas microbe is present. In some embodiments, the Pseudomonas microbe is present and is Pseudomonas nitroreducens or Pseudomonas citronellolis. In some embodiments, the Pseudomonas microbe is present and is Pseudomonas nitroreducens. In some embodiments, the Pseudomonas microbe is present and is Pseudomonas citronellolis strain ATCC 13674 or Pseudomonas citronellolis strain NRRL B-2504 or Pseudomonas citronellolis strain DSM 50332. In some embodiments, the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe.

[0009] In some embodiments, the polyethylene comprises a polyethylene selected from a group consisting of a ultra-high-molecular-weight polyethylene (UHMWPE), a ultra-low-molecular- weight polyethylene (ULMWPE or PE- WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HDPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE). In some embodiments, the polyethylene is a powder or film. In some embodiments, the polyethylene is a powder. In some embodiments, the polyethylene is a low-molecular weight polyethylene powder.

[0010] In some embodiments, the polyethylene substrate is mixed or commingled with an additive. In some embodiments, the polyethylene substrate is a mixed polyethylene plastic article. In some embodiments, the polyethylene substrate is exposed to an non-ionic surfactant. In some embodiments, the non-ionic surfactant is Triton X-100.

[0011] In some embodiments, the polyethylene is UV-pretreated. In some embodiments, the polyethylene is oxidase enzyme-pretreated. In some embodiments, the polyethylene is not UV- pretreated before combining with the Pseudomonas microbe and/or the oxidase enzyme.

[0012] In some embodiments, the oxidase enzyme is present. In some embodiments, the oxidase enzyme is present and comprises a multi-copper center structure. In some embodiments, the oxidase enzyme is present and is a laccase. In some embodiments, the oxidase enzyme is present and is a bacterial derived oxidase enzyme. In some embodiments, the oxidase enzyme is present and is a fungal derived oxidase enzyme. In some embodiments, the oxidase enzyme is present and is laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. In some embodiments, the laccase is derived from Trametes versicolor strain ATCC 42530, Trametes versicolor strain ATCC 42462, Trametes versicolor strain ATCC 20869, Trametes versicolor strain ATCC 96186, Trametes versicolor strain ATCC 42394, Trametes versicolor strain ATCC 20547, Trametes versicolor strain ATCC 11235, Trametes versicolor strain ATCC 200801, Trametes versicolor strain ATCC 48424, Trametes versicolor strain ATCC 34584, Trametes versicolor strain ATCC 58078, Trametes versicolor strain ATCC 66173, Trametes versicolor strain DSM 1977, Trametes versicolor strain DSM 3086, Trametes versicolor strain DSM 6401, Trametes versicolor strain DSM 11269, Trametes versicolor strain DSM 11309, or Trametes versicolor strain NRRL 66313. In some embodiments, the oxidase enzyme is present and is utilized to pre-treat the polyethylene, before the Pseudomonas microbe is combined with the polyethylene.

[0013] In some embodiments, the present disclosure provides both the Pseudomonas microbe and the oxidase enzyme are present. In other embodiments, the Pseudomonas microbe expresses the oxidase enzyme. In further embodiments, the polyethylene substrate’ s molecular weight is reduced by at least 10%, 20%, 30%, 40%, or 50%.

[0014] The present disclosure provides A method of degrading polyethylene, the method comprising: a) pretreating a polyethylene substrate by UV or an oxidase enzyme; and b) combining a Pseudomonas microbe with the polyethylene substrate pretreated by UV or the oxidase enzyme of step a), wherein the polyethylene substrate’s molecular weight is reduced upon a sequential exposure to (i) UV or the oxidase enzyme and (ii) the Pseudomonas microbe.

[0015] The present disclosure provides a composition comprising: a Pseudomonas microbe and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV or an oxidase enzyme. In some embodiments, a composition comprising: a Pseudomonas microbe, an oxidase enzyme, and a polyethylene substrate. In other embodiments, A composition comprising: a Pseudomonas microbe and an effective amount of a purified oxidase enzyme, wherein a polyethylene substrate is treated with the composition. In further embodiments, a composition comprising: a Pseudomonas microbe, a laccase and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV.

[0016] In some embodiments, the disclosure teaches (1) degradation of PE with a specific enzyme or analogue or variant thereof (e.g. the aforementioned laccase); (2) degradation of PE with a specific microbe (e.g. the aforementioned Pseudomonas strains); and/or (3) degradation of PE by utilizing a system/method comprising both an enzyme and microbial strain (e.g. the aforementioned laccase and Pseudomonas strains).

[0017] In embodiments, the disclosure teaches fragmentation of PE by enzymes (e.g. oxidases such as a laccase, including analogues or variants thereof) and also “bioassimilation” of PE by a microbial species (e.g. a species from the genus Pseudomonas). These processes (fragmentation and bioassimilation) can occur together/in conjunction or they can occur separately. Consequently, aspects of the disclosure are drawn to broad systems, methods, and compositions for the degradation of polymers, e.g. PE. In aspects, the polymers are natural. In other aspects, the polymers are synthetic.

BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1 illustrates 30-day time course of P. citronellolis growth on UV-pretreated Polyethylene (UV-PE) as a sole carbon source. OD600 is a measure of growth (Starting OD600 =

O.01). Three samples were tested (N=3). NA indicates no PE control ( Pseudomonas strain only). [0019] FIG. 2A illustrates change in mass between microbe cultures (i.e., P. citronellolis (P6) and

P. nitroreducens (P8)) and no strain control (NA) with 100 mg of UV-pretreated PE. FIG. 2B illustrates experimental data of mass loss in two microbial cultures, each of which contained P. citronellolis (P6) and P. nitroreducens (P8), respectively. NA indicates uninoculated control (no strain). Also, the values in FIGs. 2A-2B are statistically different (N=8).

[0020] FIG. 3A illustrates a High Temperature Gel Permeation Chromatography (HT-GPC) polymer analysis comparing a microbially-treated UV-PE (P. citronellolis ) to a microbially- untreated ETV-PE control (No strain). FIG. 3B illustrates experimental data of a weight averaged molecular weight (Mw), a number averaged molecular weight (Mn), and percentage of degradation (% degradation) between the microbially-treated ETV-PE and the microbially-untreated ETV-PE as illustrated in FIG. 3A.

[0021] FIG. 4A illustrates a High Temperature Gel Permeation Chromatography (HT-GPC) polymer analysis comparing a laccase-treated PE to a laccase-untreated PE control (No enzyme). Laccase indicates Trametes versicolor laccase enzyme and HBT indicates hydroxybenzotriazole (laccase reaction mediator). FIG. 4B illustrates experimental data of a weight averaged molecular weight (Mw), a number averaged molecular weight (Mn), and percentage of degradation (% degradation) between the laccase-treated PE and the laccase-untreated PE as illustrated in FIG. 4A.

[0022] FIG. 5 illustrates a Fourier Transform Infrared Spectroscopy (FTIR) analysis of PE with/without Laccase- mediator system (LMS) treatment for 14 days. T. versicolor laccase and HBT were incubated with PE for 14 days (LMS 14d) in comparison to no enzyme control (i.e. without T. versicolor laccase treated) and this analysis was conducted with 5 ml reaction containing 250 mg PE powder, 0.5 mM HBT, 100 mM citrate buffer (5.0), 0.05% Triton X-100, and 1 mg laccase applied 5 times over 14 days.

[0023] FIG. 6 illustrates a Fourier Transform Infrared Spectroscopy (FTIR) analysis of PE oxidation by T. versicolor laccase (14 days) compared to PE oxidation by UV irradiation (34 days). [0024] FIG. 7A illustrates 10-day cultures of P. citronellolis on PE with/without UV irradiation. Each culture was plated for CFU counting (10 ⁵ dilutions shown). FIG. 7A shows P. citronellolis growth without PE (left), P. citronellolis growth on PE without LTV irradiation (middle), and P. citronellolis growth on PE with LTV irradiation (right). FIG. 7B provides average cell counts of P. citronellolis cultures illustrated in FIG. 7A from 2 dilutions (N=2).

[0025] FIG. 8 illustrates P. citronellolis (P6) growth on four untreated PE substrates (PEI to PE4) and five UV-treated PE substrates (UV-PE1 to UV-PE4). UV-PE1 has two treatment conditions, which are UV-PEl-1 and UV-PE1-2. PEI substrate was treated with LTV for two different time periods, which are (i) UV-PEl-1: 30 days LTV treatment on PEI; (ii) UV-PE1-2: 72 days LTV treatment on PEI). NA indicates a negative control (No PE).

[0026] FIG. 9 illustrates P citronellolis growth for 8 days on (i) laccase-pretreated (20 day pretreatment) PE, (ii) untreated PE, and (iii) UV-pretreated (28-day pretreatment) PE. Starting OD600 = 0.01.

[0027] FIG. 10 illustrates P. nitroreducens growth for 7 days on (i) negative control (No PE), (ii) laccase-pretreated (20 day pretreatment) PE, and (iii) UV-pretreated (28-day pretreatment) PE. Starting OD600 = 0.01.

[0028] FIGs. 11A-11B illustrate two ways to combine enzyme sand strains for fermentation of PE. FIG. 11 A illustrates a two-step fermentation of PE by pretreating PE with laccase and fermenting the pre-treated PE, while FIG. 11B illustrates a single-step fermentation of PE with engineered bioassimilation strain in which laccases can be expressed.

[0029] FIGs. 12A-12F illustrate treatment combinations of microbe, UV, and/or oxidase for PE degradation and bioassimilation.

DETAILED DESCRIPTION OF THE DISCLOSURE

Definitions

[0030] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0031] The term “a” or “an” refers to one or more of that entity, i.e. can refer to a plural referents. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements. [0032] The term “about” as used herein with respect to % sequence identity of a nucleic acid or amino acid means up to and including ±1.0% in 0.1% increments. For example “about 90%” sequence identity includes 89.0%, 89.1%, 89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, and 91%. If not used in the context of % sequence identity, then “about” means ±10%.

[0033] As used herein, the term “allele(s)” means any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. The term “control” refers to an appropriate comparator for determining the effect of experimental treatment. In some embodiments, a control is no treatment of strain of interest (such as the microbe of the genus Pseudomonas used in the present disclosure). In other embodiments, no treatment of enzyme of interest (such as laccase used in the present disclosure). In further embodiments, a control host cell is a wild type cell or a host cell that is genetically identical to a genetically modified host cell, except for the genetic modification(s) differentiating the treatment host cell.

[0034] As used herein, the term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.

[0035] As used herein, the term “genetically linked” refers to two or more traits that are co inherited at a high rate during breeding such that they are difficult to separate through crossing. [0036] A “recombination” or “recombination event” as used herein refers to a chromosomal crossing over or independent assortment. The terms “genetically modified host cell,” “recombinant host cell,” and “recombinant strain” are used interchangeably herein and refer to host cells that have been genetically modified by the cloning and transformation methods of the present disclosure. Thus, the terms include a host cell ( e.g ., bacteria, yeast cell, fungal cell, CHO, human cell, etc.) that has been genetically altered, modified, or engineered, such that it exhibits an altered, modified, or different genotype and/or phenotype (e.g., when the genetic modification affects coding nucleic acid sequences of the microorganism), as compared to the naturally- occurring organism from which it was derived. It is understood that in some embodiments, the terms refer not only to the particular recombinant host cell in question, but also to the progeny or potential progeny of such a host cell.

[0037] The term “wild-type microorganism”, “wild-type cell” or “wild-type host cell” describes a cell that occurs in nature, i.e. a cell that has not been genetically modified. [0038] The term “genetically engineered” may refer to any manipulation of a host cell’s genome ( e.g . by insertion, deletion, mutation, or replacement of nucleic acids).

[0039] As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism, or group of organisms which results from the interaction between that individual’s genetic makeup (i.e., genotype) and the environment.

[0040] As used herein, the term “chimeric” or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid, or a protein sequence, that links at least two heterologous polynucleotides, or two heterologous polypeptides, into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence. For example, the term “recombinant” can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0041] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0042] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.

[0043] As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. [0044] As used herein, the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. For purposes of this disclosure homologous sequences are compared. “Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but are not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et ah, eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NΉ, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.

[0045] As used herein the term “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite of sequence analysis programs. Unless noted otherwise, the term “sequence identity” in the claims refers to sequence identity as calculated by ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids Res. 22:4673-4680), such as Clustal Omega® using default parameters.

[0046] As used herein, a residue (such as a nucleic acid residue or an amino acid residue) in sequence “X” is referred to as corresponding to a position or residue (such as a nucleic acid residue or an amino acid residue) “a” in a different sequence “Y” when the residue in sequence “X” is at the counterpart position of “a” in sequence “Y” when sequences X and Y are aligned using amino acid sequence alignment tools known in the art, such as, for example, Clustal Omega or BLAST®. [0047] When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Sequences which differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988). Similarity is more sensitive measure of relatedness between sequences than identity,· it takes into account not only identical (i.e. 100% conserved) residues but also non identical yet similar (in size, charge, etc.) residues. % similarity is a little tricky since its exact numerical value depends on parameters such as substitution matrix one uses (e.g. permissive BLOSUM45 vs. stringent BLOSUM90) to estimate it.

[0048] The methods and systems of the present disclosure can be used to identify sequences that are homologous/orthologous to one or more target genes/proteins or to one or more selected protein domains, or shared domains within a class of proteins of interest. In some embodiments, homologous sequences are sequences that share sequence identity with the target gene/protein (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% percent identity, including all values in between). In some embodiments, homologous sequences also carry out the same or similar biological function as the target gene/proteins.

[0049] As used herein in the terms “target protein” or “target gene” refers to a starting gene or protein (e.g., nucleic acid or amino acid sequence) for which homologs or orthologs are sought. In some embodiments, searches are conducted with more than one target gene/protein.

[0050] As used herein, the term “endogenous” or “endogenous gene,” refers to the naturally occurring gene, in the location in which it is naturally found within the host cell genome. In some embodiments, operably linking a heterologous promoter to an endogenous gene means genetically inserting a heterologous promoter sequence in front of an existing gene, in the location where that gene is naturally present. In other embodiments, an endogenous gene as described herein can include alleles of naturally occurring genes that have been mutated.

[0051] As used herein, the term “exogenous” is used interchangeably with the term “heterologous,” and refers to a substance coming from some source other than its native source. For example, the terms “exogenous protein,” or “exogenous gene” refer to a protein or gene from a non-native source or location, and that have been artificially supplied to a biological system. [0052] As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, and/or insertion, as is well understood in the art. For example, mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.

[0053] As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and/or insertion, as is well understood in the art.

[0054] As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule. A fragment of a polynucleotide of the disclosure may encode a biologically active portion of a genetic regulatory element. A biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the disclosure that comprises the genetic regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide useful as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.

[0055] Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al.( 1997) J. Mol. Biol. 272:336-347; Zhang etal.(\991) PNAS 94:4504-4509; Crameri etal.( 1998) Nature 391:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0056] For PCR amplifications of the polynucleotides disclosed herein, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3 ^rd ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

[0057] The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A/T vs. G/C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

[0058] As used herein, “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In some embodiments, the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0059] As used herein, the phrases “recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the disclosure. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones etal., (1985) EMBO J. 4:2411-2418; De Almeida etal., (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others. Vectors can be plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide- conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. As used herein, the term “expression” refers to the production of a functional end- product e.g., an mRNA or a protein (precursor or mature).

[0060] “Operably linked” means in this context the sequential arrangement of the promoter polynucleotide according to the disclosure with a further oligo- or polynucleotide, resulting in transcription of said further polynucleotide.

[0061] The term "product of interest" or "biomolecule" as used herein refers to any product produced by microbes from feedstock. In some cases, the product of interest may be a small molecule, enzyme, peptide, amino acid, organic acid, synthetic compound, fuel, alcohol, etc. For example, the product of interest or biomolecule may be any primary or secondary extracellular metabolite. The primary metabolite may be, inter alia, ethanol, citric acid, lactic acid, glutamic acid, glutamate, lysine, threonine, tryptophan and other amino acids, vitamins, polysaccharides, etc. The secondary metabolite may be, inter alia, an antibiotic compound like penicillin, or an immunosuppressant like cyclosporin A, a plant hormone like gibberellin, a statin drug like lovastatin, a fungicide like griseofulvin, etc. The product of interest or biomolecule may also be any intracellular component produced by a microbe, such as: a microbial enzyme, including: catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase, laccase, and many others. The intracellular component may also include recombinant proteins, such as: insulin, hepatitis B vaccine, interferon, granulocyte colony - stimulating factor, streptokinase and others.

[0062] The term “bioassimilation” as used herein refers to uptake and use of a substance by a microbe as a feedstock to support growth.

[0063] The term “biodegradation” as used herein refers to degradation caused by biochemical or enzymatic process resulting from the action of enzymes or microbes. In some embodiments, a substance such as a polymer (i.e. polyethylene) can be degraded by biological, biochemical, or enzymatic activity of cells or microbes. For example, polymeric substances is subject to degradation by biological, biochemical, or enzymatic activity, which results in the lowering of the molar masses of macromolecules that form the substances.

[0064] The term “carbon source” generally refers to a substance suitable to be used as a source of carbon for cell growth. Carbon sources include, but are not limited to, biomass hydrolysates, starch, sucrose, cellulose, hemicellulose, xylose, and lignin, as well as monomeric components of these substrates. Carbon sources can comprise various organic compounds in various forms, including, but are not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides such as glucose, dextrose (D-glucose), maltose, oligosaccharides, polysaccharides, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof. Photosynthetic organisms can additionally produce a carbon source as a product of photosynthesis. In some embodiments, carbon sources may be selected from polymers such as polyethylene or polythene.

[0065] The term “feedstock” is defined as a raw material or mixture of raw materials supplied to a microorganism or fermentation process from which other products can be made. In some embodiments, a carbon source, such as polymers (i.e. polyethylene) can be a feedstock for bacterial and/or microbial bioassimilation by the strains that would facilitate fermentation of the laccase- pretreated PE. In other embodiments, a carbon source, such as biomass or the carbon compounds derived from biomass are a feedstock for a microorganism that produces a product of interest ( e.g . small molecule, peptide, synthetic compound, fuel, alcohol, etc.) in a fermentation process. However, a feedstock may contain nutrients other than a carbon source.

Microbes

[0066] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms can be used interchangeably and include, but may not be limited to, the two prokaryotic domains, Bacteria and Archaea, as well as certain eukaryotic fungi and protists. In the present disclosure, “microbe,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell in its single-celled form or in a colony of cells, which is included within the domains of archaea, bacteria or eukaryota, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a substance of interest.

[0067] The term “prokaryotes” is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.

[0068] The term “Archaea” refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of ssrRNA analysis, the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt (NaCl); and extreme (hyper) thermophilus (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria (i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contains the methanogens and extreme halophiles.

[0069] “Bacteria” or “eubacteria” refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group ( Bacillus , Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas ); (2) Proteobacteria, e.g., Purple photosynthetic+non- photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6 ) Bacteroides, Flavobacteria; (7) Chlamydia, (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

[0070] In some embodiments, "gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Pseudomonas, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0071] "Gram positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces .

[0072] A “eukaryote” is any organism whose cells contain a nucleus and other organelles enclosed within membranes. Eukaryotes belong to the taxon Eukarya or Eukaryota. The defining feature that sets eukaryotic cells apart from prokaryotic cells (the aforementioned Bacteria and Archaea) is that they have membrane-bound organelles, especially the nucleus, which contains the genetic material, and is enclosed by the nuclear envelope.

[0073] In some aspects, the microbe used in the present disclosure are of bacterial strains associated with polyethylene biodegradation, which are of the genus Acinetobacter, Alcanivorax, Arthrobacter, Arthrobacter, Bacillus, Brevibacillus, Delftia, Flavobacterium, Micrococcus, Microbacterium, Nocardia, Paenibacillus, Pseudomonas, Rahnella, Ralstonia, Rhodococcus, Staphylococcus, Stenotrophomonas, Streptomyces, and Thalassolituus.

[0074] In other aspects, the microbe used in the present disclosure are of fungal strains associated with polyethylene biodegradation, which are of the genus Acremonium, Aspergillus, Chaetomium, Cladosporium, Fusarium, Glioclodium, Mortierella, Mucor, Penicillum, Phanerochaete, Trichoderma, Verticillium, and Yarrowia.

[0075] The microbe used in the present disclosure are of the genus Pseudomonas in some aspects. Pseudomonas

[0076] Pseudomonas is a genus of Gram-negative, Gammaproteobacteria, belonging to the family Pseudomonadaceae and containing at least 191 validly described species. The members of the genus demonstrate a great deal of metabolic diversity and consequently are able to colonize a wide range of niches. Due to the ease of culture in vitro and availability of an increasing number of Pseudomonas strain, genome sequences of the Pseudomonas strains have been sequenced including P. aeruginosa as a human pathogen, the plant pathogen P. syringae, the soil bacterium P. putida, and the plant growth-promoting P. fluorescens, P. lini, P. migulae and P. graminis. [0077] In some embodiments, the Pseudomonas species used in the present disclosure include but are not limited to Pseudomonas citronellolis, Pseudomonas nitroreducens, Pseudomonas protegens, Pseudomonas saponiphila, Pseudomonas ficuserectae, Pseudomonas congelans, Pseudomonas tremae, Pseudomonas caricapapayae, Pseudomonas mandelii, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas chlororaphis subsp. piscium, Pseudomonas cannabina, Pseudomonas marginalis, Pseudomonas simiae, Pseudomonas avellanae, Pseudomonas chlororaphis subsp. aurantiaca, Pseudomonas chlororaphis subsp. chlororaphis, Pseudomonas frederiksbergensis, Pseudomonas amygdali, Pseudomonas extremaustralis, Pseudomonas kilonensis, Pseudomonas lini, Pseudomonas Antarctica, Pseudomonas corrugata, Pseudomonas poae, Pseudomonas grimontii, Pseudomonas brassicacearum subsp. Neoaurantiaca, Pseudomonas meridian, Pseudomonas trivialis, Pseudomonas veronii, Pseudomonas lundensis, Pseudomonas salomonii, Pseudomonas rhodesiae, Pseudomonas arsenicoxydans, Pseudomonas thivervalensis, Pseudomonas deceptionensis, Pseudomonas palleroniana, Pseudomonas chlororaphis subsp. aureofaciens, Pseudomonas costantinii, Pseudomonas lurida, Pseudomonas migulae, Pseudomonas orientalis, Pseudomonas extremorientalis, Pseudomonas mediterranea, Pseudomonas brassicacearum subsp. brassicacearum, Pseudomonas abietaniphila, Pseudomonas baetica, Pseudomonas brenneri, Pseudomonas psychrophila, Pseudomonas jessenii, Pseudomonas fragi, Pseudomonas tolaasii, Pseudomonas proteolytica, Pseudomonas taetrolens, Pseudomonas mohnii, Pseudomonas moorei, Pseudomonas moraviensis, Pseudomonas gessardii, Pseudomonas cichorii, Pseudomonas libanensis, Pseudomonas benzenivorans, Pseudomonas panaris, Pseudomonas umsongensis, Pseudomonas reinekei, Pseudomonas fluorescens, Pseudomonas agarici, Pseudomonas lutea, Pseudomonas mucidolens, Pseudomonas azotoformans, Pseudomonas viridiflava, Pseudomonas koreensis, Pseudomonas kuykendallii, Pseudomonas synxantha, Pseudomonas segetis, Pseudomonas marincola, Pseudomonas cedrina subsp. cedrina, Pseudomonas graminis, Pseudomonas Vancouver ensis, Pseudomonas cedrina subsp. fiilgida, Pseudomonas plecoglossicida, Pseudomonas cuatrocienegasensis, Pseudomonas taiwanensis, Pseudomonas putida Pseudomonas rhizosphaerae, Pseudomonas anguilliseptica, Pseudomonas monteilii, Pseudomonas fuscovaginae, Pseudomonas mosselii, Pseudomonas taeanensis, Pseudomonas asplenii, Pseudomonas entomophila, Pseudomonas cremoricolorata, Pseudomonas parafulva, Pseudomonas alcaliphila, Pseudomonas oleovorans subsp. lubricantis, Pseudomonas borbori, Pseudomonas composti, Pseudomonas toyotomiensis, Pseudomonas batumici, Pseudomonas flavescens, Pseudomonas vranovensis, Pseudomonas punonensis, Pseudomonas balearica, Pseudomonas indoloxydans, Pseudomonas guineae, Pseudomonas japonica Pseudomonas stutzeri, Pseudomonas seleniipraecipitans, Pseudomonas peli, Pseudomonas fulva, Pseudomonas argentinensis, Pseudomonas xanthomarina, Pseudomonas pohangensis, Pseudomonas oleovorans, Pseudomonas mendocina, Pseudomonas luteola, Pseudomonas straminea, Pseudomonas caeni, Pseudomonas aeruginosa, Pseudomonas tuomuerensis, Pseudomonas azotgens, Pseudomonas indica, Pseudomonas oryzihabitans, Pseudomonas otitidis, Pseudomonas psychrotolerans, Pseudomonas zeshuii, Pseudomonas resinovorans, Pseudomonas oleovorans subsp. oleovorans, Pseudomonas thermotolerans, Pseudomonas bauzanensis, Pseudomonas duriflava, Pseudomonas pachastrellae, Pseudomonas alcaligenes, Pseudomonas xinjiangensis, Pseudomonas delhiensis, Pseudomonas sabulinigri, Pseudomonas litoralis, Pseudomonas pelagia, Pseudomonas linyingensis, Pseudomonas knackmussii, Pseudomonas panipatensis, Pseudomonas nitroreducens, Pseudomonas nitritireducens, Pseudomonas jinjuensis, Pseudomonas pertucinogena, Pseudomonas xiamenensis, Pseudomonas cissicola, Pseudomonas halophile, Pseudomonas boreopolis, Pseudomonas geniculate, Pseudomonas beteli, Pseudomonas hibiscicola, Pseudomonas pictorum, and Pseudomonas carboxydohydrogena [0078] In some embodiments, the Pseudomonas species is Pseudomonas nitroreducens or Pseudomonas citronellolis . In further embodiments, the Pseudomonas species is Pseudomonas nitroreducens. In further embodiments, the Pseudomonas species is Pseudomonas citronellolis, which is ATCC 13674, NRRL B-2504, or DSM 50332.

[0079] Microbes, such as bacteria and archaea, can be identified using their ribosomal RNA (rRNA) gene sequence, which is required for the survival of all prokaryotic microbes. Individual species of bacteria and archaea have characteristic DNA variations in the rRNA gene that serve as identifiers, fingerprints, or Unique Sequence IDentifiers (USID) for that species.

[0080] In some embodiments, Pseudomonas strains comprises a 16S rRNA sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99%, or 100% sequence identity to the 16S rRNA sequence of Pseudomonas citronellolis, which is set forth in SEQ ID NO: 1. In other embodiments, the Pseudomonas strain according to the present invention comprises a 16S rRNA sequence with at least 95% sequence identity to SEQ ID NO: 1. In further embodiments, the Pseudomonas strain according to the different embodiments of the invention comprises a 16S rRNA sequence with a at least 98% sequence identity to SEQ ID NO: 1.

[0081] Th Q Pseudomonas strains according to the different embodiments of the present invention, are characterized in that they comprises a 16S rRNA sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1.

[0082] Further, strains that have at least 95% identity to the 16S rRNA of Pseudomonas citronellolis strain or alternatively strains of which the 16S rRNA comprises a sequence with at least 95% sequence identity to the sequence of SEQ ID NO: 1 are considered “genetic equivalents”. In embodiments described and/or claimed herein, genetic equivalents may be used as an alternative in place of beneficial bacteria.

[0083] The present disclosure teaches methods of degrading polyethylene, comprising: combining a Pseudomonas microbe with a polyethylene substrate, wherein the polyethylene substrate’s molecular weight is reduced upon exposure to the Pseudomonas. In some embodiments, the Pseudomonas microbe is a microbe comprising 16S rRNA sequence with at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the Pseudomonas microbe is Pseudomonas citronellolis. In other embodiments, the Pseudomonas microbe is Pseudomonas nitroreducens. Table 1 presents 16S RNA gene sequences of exemplary Pseudomonas strains; Pseudomonas citronellolis, Pseudomonas nitroreducens, and Pseudomonas putida.

[0084] Table 1. 16S RNA gene sequences of exemplary Pseudomonas strains

Enzymes

[0085] The term "enzyme" as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.

Oxidase

[0086] An oxidase is an enzyme that catalyzes an oxidation-reduction reaction, especially one involving dioxygen (O2) as the electron acceptor. In reactions involving donation of a hydrogen atom, oxygen is reduced to water (H2O) or hydrogen peroxide (H2O2). The oxidases are a subclass of the oxidoreductases, and the exemplary lists of oxidase are as follows: cytochrome c oxidase, glucose oxidase, Monoamine oxidase, cytochrome P450 oxidase, NADPH oxidase, Xanthine oxidase, L-gulonolactone oxidase, Laccase, Lysyl Oxidase, Polyphenol oxidase, and Sulfhydryl oxidase.

[0087] Laccaseis multi-copper oxidase found in plants, fungi, lichen, insects and bacteria, which contain 15-30% carbohydrate and have a molecule mass of 60-90 kDa. Laccase, which belongs to the enzyme family of multi-copper oxidases (MCOs), is classified as benzenediol oxygen reductase (EC 1.10.3.2) and are also known as urushiol oxidases and p-diphenol oxidases. They are considered versatile enzymes capable of oxidizing a large number of phenolic and non- phenolic molecules due to their low substrate specificity, using oxygen as electron acceptor and generating water as a by-product (Kim C, Lorenz WW, Hoopes JT, Dean JF. J Bacteriol. 2001;183:4866-75; Morozova OV et al, Appl Biochem Microbiol. 2007;43:523-35; Upadhyay P et al, 2016;3 Biotech 6, 15). UniProtKB search results for “laccase” with sequence sizes between 220 and 800 amino acids, revealed approximately 7300 cellular-organism sources, with 1026 bacteria, 6258 eukaryotes, and 16 halobacteria (archaea). Hence, it can be predicted that this large number of enzymes produced by different organisms could have a wide range of applications in water bioremediation (Arregui, L., Ayala, M, Gomez-Gil, X. et al. 2019; Microb Cell Fact 18,

200)

[0088] Laccase participates in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. In some embodiments, laccase can be used in the design of biosensors, biofuel cells, as a medical diagnostics tool and bioremediation agent to clean up herbicides, pesticides and certain explosives in soil due to their ability to oxidize both phenolic and nonphenolic lignin-related compounds as well as highly recalcitrant environmental pollutants. [0089] In some embodiments, enzymatic actions of laccase are associated with sporulation, detoxification, morphogenesis, melanin polymerization and it offers protection to spore coat. In some embodiments, laccase utilizes oxygen for its catalysis, which is useful in the biological degradation of micropollutants in wastewater treatment. In some embodiments, laccase catalyzes the oxidation of phenol containing compounds, including lignin, through the reduction of oxygen to water. In other embodiments, the presence of mediators will allow the oxidation of non-phenlic compounds as well.

[0090] The present disclosure teaches that laccase can be used to catalyze oxidation of polymers such as polyethylene and reduce molecular weight of the polymers, thereby facilitating degradation of the polymers.

[0091] Laccase is generally found in higher plants and fungi, as well as in some bacteria such as S.lavendulae, S.cyaneus, and Marinomonas mediterranea. In fungi, laccases appear more than the higher plants. Basidiomycetes such as Phanerochaete chrysosporium, Theiophora terrestris, and Lenzites, betulina, and white-rot fungi such as Phlebia radiate, Pleurotus ostreatus, and Trametes versicolour also produce laccase.

[0092] In some embodiments, the oxidase is laccase.

[0093] In some embodiments, the oxidase comprises multi-copper center structure.

[0094] In some embodiments, the oxidase is a bacterial derived oxidase enzyme.

[0095] In some embodiments, the oxidase is a fungal derived oxidase enzyme.

[0096] In some embodiments, the oxidase is laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor, Trametes villosa, or Thielavia arenaria.

[0097] Table 2 presents Laccase sequences (EC 1.10.3.2) from Trametes species and Table 3 from Thielavia Arenaria.

[0098] Table 2. Laccase sequences (EC 1.10.3.2) from Trametes species

[0099] Table 3. Laccase sequence (EC 1.10.3.2) from Thielavia arenaria

[0100] In some embodiments, laccase is present in the genus Trametes. In some embodiments, laccase is present in Trametes versicolor (T. versicolor). In some embodiments, laccase is present in T. versicolor strain, which is ATCC 42530, ATCC 42462, ATCC 20869, ATCC 96186, ATCC 42394, ATCC 20547, ATCC 11235, ATCC 200801, ATCC 48424, ATCC 34584, ATCC 58078, ATCC 66173; DSM 1977, DSM 3086, DSM 6401, DSM 11269, DSM 11309; and NRRL 66313. [0101] In other embodiments, laccase is derived from T. versicolor strain, which is ATCC 42530, ATCC 42462, ATCC 20869, ATCC 96186, ATCC 42394, ATCC 20547, ATCC 11235, ATCC 200801, ATCC 48424, ATCC 34584, ATCC 58078, ATCC 66173; DSM 1977, DSM 3086, DSM 6401, DSM 11269, DSM 11309; and NRRL 66313.

[0102] In some embodiments, the laccase derived from T. versicolor strain, which is ATCC 42530, ATCC 42462, ATCC 20869, ATCC 96186, ATCC 42394, ATCC 20547, ATCC 11235, ATCC 200801, ATCC 48424, ATCC 34584, ATCC 58078, ATCC 66173; DSM 1977, DSM 3086, DSM 6401, DSM 11269, DSM 11309; and NRRL 66313 is present and the polyethylene is degraded or assimilated by the laccase. In further embodiments, laccase can reduce the molecular weight of PE powder.

[0103] In some embodiments, laccase is purified from Agaricus bisporus, Aspergillus sp., Rhus vemicifera or Trametes versicolor. In some embodiments, laccase is present in a purified powder form derived from Agaricus bisporus, Aspergillus sp., Rhus vemicifera or Trametes versicolor. In some embodiments, laccase is purified from T. versicolor. In further embodiments, laccase is present in a purified powder form derived from T. versicolor.

[0104] In further embodiments, the enzyme taught herein (i.e. laccase) may be in soluble form, or on solid phase such as powder form. It may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic, polymers, filter, membranes, e.g., in the form of beads, columns, plates and the like. The enzyme may be in an isolated or purified form. In some embodiments, the enzymes of the present disclosure are expressed, derived, secreted, isolated, or purified from microorganisms. The enzymes may be purified by techniques known per se in the art, and stored under conventional techniques. The enzymes may be further modified to improve e.g., their stability, activity and/or adsorption on the polymer. For instance, the enzymes are formulated with stabilizing and/or solubilizing components, such as water, glycerol, sorbitol, dextrin, including maltodextrine and/or cyclodextrine, starch, propanediol, salt, etc.

Nucleic Acid Molecules Encoding Laccase Proteins

[0105] One aspect of the disclosure pertains to isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding laccase polypeptides, proteins, or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology.

[0106] As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded.

[0107] An “isolated” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in vitro. A “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or fungal host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences that naturally flank the nucleic acid i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecule encoding a laccase protein of the disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0108] In some embodiments, an isolated nucleic acid molecule encoding a laccase protein has one or more changes in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments, the change in the native or genomic nucleic acid sequence includes, but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion, and/or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding a laccase protein is a non-genomic sequence. [0109] A variety of polynucleotides that encode a laccase protein of the disclosure are contemplated. Such polynucleotides are useful for production of the laccase proteins in host cells when operably linked to suitable promoter, transcription termination and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode further laccase proteins.

[0110] Polynucleotides that encode a laccase protein of the disclosure can be synthesized de novo from a sequence disclosed herein. The sequence of the polynucleotide gene can be deduced from a disclosed protein sequence through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide.

[0111] Furthermore, synthetic polynucleotide sequences of the disclosure can be designed so that they will be expressed in bacteria or fungi. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0112] In some embodiments, a nucleic acid molecule encoding laccase protein of the disclosure is a non-genomic nucleic acid sequence. As used herein a “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” refers to a nucleic acid molecule that has one or more changes in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments, the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron associated with the genomic nucleic acid sequence; insertion of one or more heterologous intrans; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of a polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a cDNA.

[0113] In some embodiments, the disclosure teaches nucleic acid molecules that encode laccase proteins taught herein, as well as nucleic acid molecules that encode proteins taught herein that have had an amino acid substitution, deletion, insertion, and fragments thereof and combinations thereof.

[0114] Also provided are nucleic acid molecules that encode transcription and/or translation products that are subsequently spliced to ultimately produce functional laccase proteins. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing. The substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a laccase protein encoding sequence. An example of trans-splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full- length pesticidal protein encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides (U.S. Pat. Nos. 6,365, 377 and 6,531,316). Thus, in some embodiments the polynucleotides do not directly encode a full-length laccase protein, but rather encode a fragment or fragments of same. [0115] Nucleic acid molecules that are fragments of the aforementioned sequences encoding laccase proteins are also encompassed by the embodiments. “Fragment” as used herein refers to a portion of the nucleic acid sequence encoding a laccase protein. A fragment of a nucleic acid sequence may encode a biologically active portion of a protein or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed herein. Nucleic acid molecules that are fragments of a nucleic acid sequence comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or more contiguous nucleotides, or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a laccase protein taught herein. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the laccase protein. In some embodiments, a fragment of a nucleic acid sequence will encode at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

290, 300, or more contiguous amino acids, or up to the total number of amino acids present in a full-length laccase protein taught herein. In some embodiments, the fragment is an N-terminal and/or a C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids from the N-terminus and/or C-terminus relative to a laccase protein taught herein, e.g., by proteolysis, insertion of a start codon, deletion of the codons encoding the deleted amino acids with the concomitant insertion of a stop codon or by insertion of a stop codon in the coding sequence.

[0116] In some embodiments, a laccase protein is sufficiently similar to the amino acid sequence of SEQ ID NOs:4-35. In other embodiments, a laccase protein is sufficiently similar to the amino acid sequence of SEQ ID NOs:4-35 as presented in Table 2. “Sufficiently similar” is used herein to refer to an amino acid sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity/similarity compared to a reference sequence using one of the alignment programs described herein, or known to one of skill in the art, using standard parameters.

[0117] In some embodiments, a laccase protein has an amino acid sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:4-35.

[0118] Table 4. Nucleic Acid Sequences encoding laccase from Trametes versicolor and Thielavia arenaria

[0119] In some embodiments, a laccase protein is encoded by a nucleic acid sequence that has sufficient sequence identity to the nucleic acid sequence of SEQ ID NOs:36-41. In some embodiments, a laccase protein is encoded by a nucleic acid sequence that has sufficient sequence identity to the nucleic acid sequence of SEQ ID NOs:36-41 as presented in Table 4. In some embodiments, a laccase protein is encoded by a nucleic acid sequence that has sufficient sequence identity to the nucleic acid sequence of SEQ ID NOs:36-41. “Sufficient sequence identity” is used herein to refer to an amino acid or nucleic acid sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity/similarity compared to a reference sequence using one of the alignment programs described herein, or known to one of skill in the art, using standard parameters.

[0120] In some embodiments, a laccase protein is encoded by a nucleic acid sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40.

[0121] In some embodiments, a laccase protein is encoded by a nucleic acid sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

Nucleotide Constructs, Expression Cassettes, and Vectors

[0122] The use of the term “nucleotide constructs” herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences, which can be employed in the methods of the embodiments for transforming plants including, but are not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but are not limited to, single-stranded forms, double-stranded forms, hairpins, stem- and-loop structures and the like. [0123] A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, fungi, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.

[0124] The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term “operably linked” also refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and were necessary to join two protein-coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0125] In some embodiments, the DNA construct comprises a polynucleotide encoding a laccase protein taught herein, which is operably linked to an endogenous regulatory sequence or a heterologous regulatory sequence.

[0126] Such a DNA construct is provided with a plurality of restriction sites for insertion of the polypeptide gene sequence to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0127] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host, e.g. a bacterial cell, a fungal cell or plant cell.

[0128] The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign, or heterologous to the host organism and/or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is “heterologous” to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments (i.e., not the native location). As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0129] In some embodiments, the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, intrans with gene expression enhancing properties in plants (US Patent Application Publication Number 2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464; Christensen and Quail (1996) Transgenic Res. 5:213-218; Christensen et al. (1992) Plant Molecular Biology 18:675-689)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al, (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9: 1685-96), the maize Adhl intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48; Kyozuka et al. (1990) Maydica 35:353-357), the enhancers of U.S. Pat. No. 7,803,992, and the sugarcane bacilliform viral (SCBV) enhancer of W02013130813 may also be used, each of which is incorporated by reference. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments. [0130] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the host or any combination thereof).

[0131] Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is bacteria, fungi, or plants, the synthetic nucleic acids can be synthesized using host-preferred codons for improved expression.

[0132] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon like repeats, and other well characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell that contains a vector and supports the replication and/or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as fungi, yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0133] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (ElroyStein, et al., (1989) Proc. Natl. Acad. Sci. EISA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al, (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMY RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968.

[0134] Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate co- translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus. “Signal sequence” as used herein refers to a sequence that is known or suspected to result in cotranslational or post- translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. “Leader sequence” as used herein refers to any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Nuclear encoded proteins targeted to the chloroplast thylakoid lumen compartment have a characteristic bipartite transit peptide, composed of a stromal targeting signal peptide and a lumen targeting signal peptide. The stromal targeting information is in the amino-proximal portion of the transit peptide. The lumen targeting signal peptide is in the carboxyl-proximal portion of the transit peptide, and contains all the information for targeting to the lumen.

[0135] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

Polyethylene

[0136] A “polymer” refers to a chemical compound or mixture of compounds whose structure is constituted of multiple repeating units (i.e. “monomers”) linked by covalent chemical bonds. Within the context of the invention, the term “polymer” includes natural or synthetic polymers, comprising a single type of repeating unit (i.e., homopolymers) or different types of repeating units (i.e., block copolymers and random copolymers). As an example, synthetic polymers include polymers derived from petroleum oil, such as polyolefins, aliphatic or aromatic polyesters, polyamides, polyurethanes and polyvinyl chloride. Natural polymers include lignin, polysaccharides, such as cellulose, hemi-cellulose, starch, and polyhydroxyalkanoates and derivatives thereof.

[0137] In another embodiment, the terms “polyethylene” and “plastic” are used interchangeably. In another embodiment, plastic is polyester, polyurethane, polypropylene, polyvinyl chloride, nylon, polystyrene, starch, and any combination thereof. In another embodiment, polystyrene includes poly butylene succinate (PBS), poly butylsuccinate adipate (PBSA), poly lactic acid (PLA), aliphatic polyester, polycaprolactone and any combination thereof. In another embodiment, plastic is biodegradable plastic. In another embodiment, biodegradable plastic is plastic that keeps its function during a use state and will be degraded to a simpler molecular level by the function of the composition of the invention. In another embodiment, plastic to be degraded may take any form such as emulsion and solid pellet depending the type of degradation reaction. In another embodiment, polyethylene is treated by thermo-oxidation. In another embodiment, polyethylene is treated by oxidation. In another embodiment, polyethylene is treated by both thermo-oxidation and oxidation. [0138] In the course of this degradation procedure, the molecular weight of the polymer is reduced to such an extent that products formed therefrom are rapidly degraded to form monomers and are completely decomposed. This applies in particular to films, sheet-like products, coatings, adhesives, injection- molded parts and granular materials made of biodegradable polymers.

[0139] Polyethylene or polythene (PE) is the most common plastic. Its primary use is in packaging (plastic bags, plastic films, geomembranes, containers including bottles, etc.). Many kinds of polyethylene are known, with most having the chemical formula (C2H4)n. PE is usually a mixture of similar polymers of ethylene with various values of n. Polyethylene is a thermoplastic; however, it can become a thermoset plastic when modified (such as cross-linked polyethylene).

[0140] The most common polyethylene types are: Low Density Polyethylene (LDPE), High Density Polyethylene (HDPE), Linear Low Density Polyethylene (LLDPE) and Cross Linked Polyethylene (XLPE). They differ in their density, degree of branching and availability of functional groups on the surface.

[0141] In some embodiments, the polyethylene substrate is selected from a group consisting of a ultra-high-molecular-weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high- density polyethylene (HDPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE)

[0142] Polyethylene is known for being a remarkably resistant polymer to degradation. Its chemical and biological inertness has fostered its application into various products from plastic bags and piping to the construction of fuel storage tanks. In some embodiments, degradation of polyethylene can be classified as abiotic or biotic. In some embodiments, the abiotic degradation of PE can defined as deterioration caused by environmental factors such as temperature, UV irradiation, while the biotic degradation of PE can be defined as biodegradation caused by the action of microorganisms that modify and consume the polymer leading to changes in its properties.

[0143] The present disclosure teaches that microbes able to colonize the surfaces of polyethylene have diverse effects/changes on properties of PE such as functional groups on the surface, hydrophobicity and/or hydrophilicity, crystallinity, surface topography, mechanical properties, molecular weight distribution and mass balance in order to establish the extent of biodegradation of the polymer.

[0144] In some embodiments, the polyethylene is a low-molecular weight polyethylene. In some embodiments, the polyethylene is a powder or film. In some embodiments, the polyethylene is a powder. In some embodiments, the polyethylene is a low-molecular weight polyethylene powder. In some embodiments, the polyethylene is UV-treated. In some embodiments, the polyethylene is enzyme-treated. In some embodiments, the polyethylene is not UY-treated before combining with the Pseudomonas microbe and/or the oxidase enzyme.

[0145] There are numerous aspects of the present disclosure that are not taught in the art, with respect to Pseudomonas species growing on and/or degrading PE (Bhatia et al, 2014; Montazer et al, 2019; Montazer et al., 2018; Kyaw et al, 2012). Among others, consider: First, degradation of PE by P. nitroreducens has not been previously described. Second, the specific strain P. citronellolis ATCC 13674 (= NRRL B-2504 = DSM 50332) has not ever been shown to degrade PE. Third, neither of these two species has been shown to grow on, or degrade, a PE powder. The prior literature report of a wild isolate of P. citronellolis degrading PE used a PE sheet and measured % weight loss of the sheet (Bhatia et al, 2014). PE sheets or films typically contain additives such as plasticizers colorants, stabilizers (e.g. antioxidants), nucleators, and clarifiers and in some cases starch-based catalysts to promote biodegradation. In some embodiments, a plastic article, product or waste comprises polyethylene (PE) with additives including, but are not limited to, plasticizers, mineral or organic fillers, oxygen scavengers, compatibilizers, adhesives, inks, colorants, stabilizers, nucleators, clarifiers, lubricants, blowing agents, light stabilizers, nucleating agents, antistatic agents, destabilizes and/or antioxidants.

[0146] The present disclosure teaches that the PE containing additives described herein is a substrate for degradation and/or bioassimilation by an action/activity of a microbe or a plurality of microbes described herein. In other embodiments, the PE containing additives described herein is a substrate for degradation and/or bioassimilation by an action/activity of a microbe or a plurality of microbes described herein in combination with UV and/or oxidase treatment.

[0147] The present disclosure uses P. citronellolis and P. nitroreducens to degrade a low- molecular weight PE powder, as well as other low molecular weight PE powders. Fourth, while % weight loss measurements have been reported previously from Pseudomonas species degrading PE, no Pseudomonas species has ever been shown to reduce the molecular weight of any PE material. The present disclosure reports a reduction in molecular weight by P. citronellolis ( P . nitroreducens has yet to be tested in this manner, but is suspected to perform similarly). Finally, the present disclosure includes a method for cultivating Pseudomonas nitroreducens and Pseudomonas citronellolis that facilitates growth on PE.

[0148] Regarding enzymatic degradation of PE, a laccase from Trametes versicolor has previously been shown to degrade PE membranes (Fujisawa et al, 2002), but the present disclosure shows this enzyme can reduce the molecular weight of pure PE powder. This powder degraded by the present disclosure is low-molecular weight PE powder. The present disclosure utilizes an oxidative degradation mechanism, which was not previously shown in literature (Fujisawa et al, 2002). [0149] Regarding combining enzymatic and microbial degradation technologies, the present disclosure uses an enzyme (laccase) to pre-treat the PE to speed microbial biodegradation or bioassimilation. A number of literature sources show enhanced microbial degradation of PE that has been oxidatively degraded, either naturally or artificially. The present disclosure overcomes the need to use a UV source or natural weathering processes and instead uses a laccase enzyme to pre-treat the PE, in order to enhance microbial biodegradation or bioassimilation. The result is an end-to-end PE degradation process that is entirely biological in nature.

[0150] In aspects, the microbial strain degrades PE. In aspects, the enzyme oxidatively degrades PE. In aspects, the PE substrate is degraded by either a microbe or an enzyme. In aspects, the mediator enables oxidation of PE by laccase. In aspects, the method for detecting growth shows growth of microbial strains using PE. In aspects, the method for recovering PE reveals any mass loss after biological treatment and allows further analysis of the purified product. In aspects, the method for analyzing molecular weight reveals any molecular weight changes after biological treatment.

[0151] In aspects, the microbial strain interacts with the PE substrate by degrading it and using portions of it to support growth. The microbial strain interacts with the method for detecting growth by releasing DNA during sample preparation to be quantified as a measure of biomass production. The microbial strain interacts with the method for recovering PE by dissolving and being removed from the PE.

[0152] In aspects, the enzyme interacts with the PE substrate by oxidatively degrading it. The enzyme interacts with the mediator by oxidizing the mediator. The enzyme interacts with the method for recovering PE by being removed from the PE. [0153] In aspects, the PE substrate interacts with the microbial strain by supporting its growth via its degradation and assimilation. The PE substrate interacts with the enzyme by becoming oxidized by the reaction the enzyme initiates. The PE substrate interacts with the mediator by becoming directly oxidized by the mediator. The PE substrate interacts with the method for recovering PE by becoming purified of biological contaminants. The PE substrate interacts with the method for analyzing molecular weight by having its molecular weight chromatographically observed relative to a standard curve.

[0154] In aspects, the mediator interacts with the enzyme by becoming oxidized by the enzyme. The mediator interacts with the PE substrate by oxidizing the PE substrate.

[0155] In aspects, the method for detecting growth interacts with the microbial strain by measuring its DNA content in a culture.

[0156] In aspects, the method for recovering PE interacts with the microbial strain by removing it from the PE. The method for recovering PE interacts with the enzyme by removing it from the PE. The method for recovering PE interacts with the PE by purifying it from either microbial culture or enzyme assay. The method for recovering PE interacts with the method for analyzing molecular weight by preparing a PE sample for analysis via the molecular weight method.

[0157] In aspects, the method for analyzing molecular weight interacts with the PE substrate by allowing observation of the PE’s molecular weight relative to a standard curve. The method for analyzing molecular weight interacts with the method for recovering PE by using the recovered PE samples for the molecular weight analysis.

Composition

[0158] The present disclosure teaches a composition comprising a Pseudomonas microbe and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV or an oxidase enzyme. The oxidase enzyme is a laccase.

[0159] The present disclosure provides a composition comprising a Pseudomonas microbe, an oxidase enzyme, and a polyethylene substrate. The oxidase enzyme is a laccase.

[0160] Provided herein is a composition comprising a Pseudomonas microbe and an effective amount of a purified oxidase enzyme, wherein a polyethylene substrate is treated with the composition. The oxidase enzyme is a laccase.

[0161] In some embodiments, a composition comprising: a Pseudomonas microbe, a laccase and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV. [0162] In some embodiments, the polyethylene substrate’s molecular weight is reduced upon exposure to the Pseudomonas microbe and/or the oxidase enzyme. In some embodiments, the Pseudomonas microbe is present and is Pseudomonas nitroreducens or Pseudomonas citronellolis . [0163] In some embodiments, the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe. The present disclosure teaches that PE can be utilized as a carbon source for bio-utilization.

[0164] In some embodiments, the polyethylene substrate is a powder or film. The polyethylene substrate is a low-molecular weight polyethylene powder.

[0165] According to the composition of the present disclosure, the polyethylene substrate is UV- pretreated or enzyme-pretreated/laccase-pretreated. In other embodiments, the polyethylene substrate is not UV-pretreated before combining with the Pseudomonas microbe and/or the oxidase enzyme.

[0166] According to the composition of the present disclosure, the oxidase enzyme is present and is utilized to pre-treat the polyethylene, before the Pseudomonas microbe is combined with the polyethylene. Both the Pseudomonas microbe and the oxidase enzyme are present. The Pseudomonas microbe expresses the oxidase enzyme. The polyethylene substrate’s molecular weight is reduced by at least 10%, 20%, 30%, 40%, or 50%.

Method for degrading polymers

[0167] A “degrading process” in relation to a plastic article refers to a process by which at least one polymer of plastic article is degraded in smaller molecules, such as monomers, oligomers, water and/or carbon dioxide.

[0168] The present disclosure teaches the degrading process that comprises a step of depolymerization. In some embodiments, the depolymerizing step may comprise a chemical depolymerization and/or a biological depolymerization.

[0169] In other embodiments, the degrading process of the disclosure comprises contacting or combining the polymers (i.e polyethylene) with a microbe and/or an oxidase enzyme (i.e., a laccase). In further embodiments, the microbe may express or comprise a depolymerase that is able to degrade at least one polymer that has been treated by UV or a laccase.

[0170] The present disclosure provides a method of degrading polyethylene, comprising: combining a Pseudomonas microbe and/or an oxidase enzyme with a polyethylene substrate. In some embodiments, the polyethylene substrate’s molecular weight is reduced upon exposure to the Pseudomonas microbe and/or the oxidase enzyme. In some embodiments, the Pseudomonas microbe is present and is Pseudomonas nitroreducens or Pseudomonas citronellolis .

[0171] In some embodiments, the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe. The present disclosure teaches that PE can be utilized as a carbon source for bio-utilization.

[0172] In some embodiments, the polyethylene substrate is a powder or film. The polyethylene substrate is a low-molecular weight polyethylene powder.

[0173] In some embodiments, the polyethylene substrate is a blown film. The polyethylene substrate is a low-molecular weight polyethylene powder.

[0174] In some embodiments, the polyethylene substrate is mixed or commingled with other plastic materials. In some embodiments, the polyethylene substrate is a mixed polyethylene plastic article. As used herein, the term “mixed polyethylene plastic article” or “mixed PE plastic article” refers to any item or product or material (such as plastic sheet, film, tube, rod, profile, shape, massive block, fiber, etc.) comprising polyethylene (PE) and at least one additional component. In some embodiments, a mixed PE plastic article encompasses all kind of plastic articles composed of polyethylene (PE) and further component(s) arranged relative to each other in such a way that they cannot be easily separated. For example, the mixed PE plastic article is a manufactured product like packaging, agricultural films, disposable items or the like, carpet scrap, fabrics, textiles etc. Furthermore, the polymers of the mixed PE plastic article may be crystallized and/or semi-crystallized polymers or amorphous polymers or a mix of crystallized and/or semi- crystallized and amorphous polymers.

[0175] In further embodiments, the method or process for PE degradation of the present disclosure can be applied to mixed PE plastic articles. The mixed PE plastic articles can come directly from plastic waste collection and/or post-industrial waste. Also, the method or process for PE degradation of the present disclosure can be applied to a mix of plastic wastes, including plastic bottles, plastic bags and plastic packaging, soft and/or hard plastics, even polluted with food residues, surfactants, etc. According to the disclosure, the additional component(s) of the mixed PE plastic article can be selected from polymers, such as polyesters, polyamides, polyolefins or vinyl polymers, metal compounds, glass compounds, fibers, paper, minerals, wood or wood compounds such as lignin, cellulose or hemi-cellulose, and starch and derivatives thereof. [0176] The present disclosure teaches that the mixed PE plastic articles are substrates for degradation and/or bioassimilation by an action/activity of a microbe or a plurality of microbes described herein. In other embodiments, the mixed PE plastic articles are substrates for degradation and/or bioassimilation by an action/activity of a microbe or a plurality of microbes described herein in combination with UV and/or oxidase treatment.

[0177] Furthermore, the mixed PE plastic article may contains, in addition to the additional component(s), other substances or additives, such as plasticizers, mineral or organic fillers, oxygen scavengers, compatibilizers, adhesives, inks, colorants, stabilizers, nucleators, clarifiers, lubricants, blowing agents, light stabilizers, nucleating agents, antistatic agents, destabilizes and/or antioxidants. In some embodiments, the polyethylene substrate is mixed or commingled with additives described herein, which is also subject to the method or process for PE degradation of the present disclosure.

[0178] According to the method or process of the present disclosure, non-ionic surfactant can be mixed or contacted with a PE substrate or a mixed PE plastic article. In some embodiments, the method or process for PE degradation of the present disclosure further comprises exposing PE substrates taught herein to at least one non-ionic surfactant. In some embodiments, the compositions of the disclosure further comprise at least one non-ionic surfactant. As used herein, the term “surfactant” is wetting agents that lower the surface tension of a substance and/or lower the interfacial tension between two substances. A surfactant that does not have a positive or negative charge in water, yet is soluble in water, is a “non-ionic surfactant”. In some embodiments, at least one surfactant is used. In other embodiments, at least one surfactant used herein is a non ionic surfactant.

[0179] In some embodiments, the surfactant is a detergent. Examples of suitable non-ionic surfactants include, but are not limited to: the “Triton” series of detergents (such as, Triton X-100 (t-Octylphenoxypoly ethoxy ethanol) and its derivatives, Triton X-114, Triton X-405, Triton X- 101, Triton N-42, Triton N-57, Triton N-60, Triton X-15, Triton X-35, Triton X-45, Triton X-102, Triton X-155, Triton X-165, Triton X-207, Triton X-305, Triton X-705-70 and Triton B-1956), the “Tween” series of detergents); Sorbitan esters; Nonylphenyl Polyethylene Glycol; the Air Products series of Surfynol surfactants; and Octylphenol Ethoxylate surfactant.

[0180] In one embodiment, the non-ionic surfactant is Triton X-100, which can be used to disperse the PE substrates in liquid. The present disclosure teaches that Triton X-100 may reduce the hydrophobicity of the PE, thereby promoting interaction of PE substrates (e.g., biofilm formation) with microbes.

[0181] According to the method of the present disclosure, the polyethylene substrate is UV- pretreated or enzyme-pretreated/laccase-pretreated. In other embodiments, the polyethylene substrate is not UV-pretreated before combining with the Pseudomonas microbe and/or the oxidase enzyme.

[0182] According to the method of the present disclosure, the oxidase enzyme is present and is utilized to pre-treat the polyethylene, before the Pseudomonas microbe is combined with the polyethylene. Both the Pseudomonas microbe and the oxidase enzyme are present. The Pseudomonas microbe expresses the oxidase enzyme. The polyethylene substrate’s molecular weight is reduced by at least 10%, 20%, 30%, 40%, or 50%.

[0183] The present disclosure teaches possible combinations of concurrent or sequential treatments of microbe, UV, and/or oxidase for PE degradation and bioassimilation as presented in

FIGs. 12A-12F.

Recombinant microorganisms

[0184] In some embodiments, the plastic product comprising polyethylene is contacted or combined with a microorganism that expresses and/or excretes an enzyme of interest (such as oxidase enzyme taught in the disclosure. In the context of the disclosure, the enzyme may be excreted in the culture medium or towards the cell membrane of the microorganism wherein said enzyme may be anchored. Said microorganism may naturally synthesize the enzyme of interest, or it may be a recombinant microorganism, wherein a recombinant nucleotide sequence encoding the oxidase enzyme (i.e. laccase) has been inserted, using a vector. For example, a nucleotide molecule, encoding the laccase of interest is inserted into a vector, e.g. plasmid, recombinant virus, phage, episome, artificial chromosome, and the like. Transformation of the host cell as well as culture conditions suitable for the host are well known to those skilled in the art.

[0185] The recombinant microorganisms may be used directly. Alternatively, or in addition, recombinant enzymes may be purified from the culture medium. Any commonly used separation/purification means, such as salting-out, gel filtration, hydrophobic interaction chromatography, affinity chromatography or ion exchange chromatography may be used for this purpose. In some embodiments, microorganisms known to synthesize and excrete laccases of interest may be used. [0186] According to the disclosure, several microorganisms and/or purified enzymes and/or synthetic enzymes may be used together or sequentially to depolymerize different kinds of polymers contained in a same plastic product or in different plastic products.

[0187] The microorganism of the disclosure exhibits a modified metabolism in order to prevent the consumption of the monomers and/or oligomers obtained from the degraded polymers. For example, the microorganism is a recombinant microorganism, wherein the enzymes degrading said monomers and/or oligomers have been deleted or knocked out. Alternatively, the process of the disclosure may be performed in a culture medium containing at least one carbon source usable by the microorganism so that said microorganism consumes this carbon source instead of the monomers and/or oligomers.

[0188] In some embodiments, the plastic product is contacted or combined with a culture medium containing the microorganisms, glucose or the like as a carbon source, as well as an available nitrogen source, including an organic nitrogen source (e.g., peptone, meat extract, yeast extract, corn steep liquor) or an inorganic nitrogen source (e.g., ammonium sulfate, ammonium chloride). The culture medium may further contain inorganic salts (e.g., sodium ion, potassium ion, calcium ion, magnesium ion, sulfate ion, chlorine ion, phosphate ion). Moreover, the medium may also be supplemented with trace components such as vitamins and amino acids.

[0189] In other embodiments, the laccase is used under conditions favoring its adsorption on the plastic product, so that the polymer of the plastic article is more efficiently oxidized or depolymerized up to monomers and/or oligomers. More particularly, the laccase may be a mutated enzyme having improved affinity for the polymer of the plastic product compared to a wild-type enzyme. Alternatively, the laccase may be used with plastic-binding proteins or binding modules that enhance the binding between the laccase and the polymer of the plastic product.

[0190] The time required for oxidation or depolymerization of at least one polymer of the plastic product may vary depending on the plastic product and its polymer itself (i.e., nature and origin of the plastic product, its composition, shape, molecular weight, etc.), the type and amount of microorganisms/enzymes used, as well as various process parameters (i.e., temperature, pH, additional agents, UV treatment etc.). The temperature is maintained below an inactivating temperature, which corresponds to the temperature at which the enzyme of interest is inactivated and/or the recombinant microorganism does no more synthesize the enzyme of interest. EXAMPLES

[0191] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.

EXAMPLE 1: Pseudomonas strains grow on polyethylene (PE) as sole carbon source [0192] To identify whether microbial strains can use PE as a carbon source for their growth, microbial strains were tested for 30-day time course growth on PE. Among them, two strains were identified and able to grow on PE as the carbon source, which were (i) Pseudomonas citronellolis and (2) Pseudomonas nitroreducens . FIG. 1 shows 30-day time course of P. citronellolis growth on UV-pretreated PE (UV-PE) compared to a P. citronellolis control without PE (NA = no PE control), indicating that Pseudomonas strains grow on PE as a sole carbon source.

[0193] The results confirm that the Pseudomonas strains can assimilate PE and naturally convert PE to biomass and metabolites. Also, growth rates of the strains are faster on UV-treated PE compared to UV-untreated PE.

EXAMPLE 2: Pseudomonas strains assimilate polyethylene

[0194] To test any change in PE mass after 30-day microbial cultures, the inventors selected two Pseudomonas strains that showed growth on PE in the previous example; Pseudomonas citronellolis (P6) and Pseudomonas nitroreducens (P8). At first, 100 mg of ETV-treated PE (ETV- PE) was put in a shake flask with each selected strain. After 30 days, the PE powder was recovered from the flasks, cleaned of all biomass, and weighed.

[0195] In this experiment, about 2 mg of PE mass loss was observed from each culture of the selected strains as presented in FIGs. 2A-2B. The results confirm that each strain assimilated ~2% of the 100 mg of the starting UV-treated PE. As the 30-day microbial cultures showed a ~2% mass loss in the PE, this indicates the Pseudomonas strains assimilated PE.

EXAMPLE 3: Pseudomonas strains assimilate polyethylene and lead to a 25% reduction in molecular weight of polyethylene

[0196] To test any reduction in molecular weight of UV-treated PE in the selected Pseudomonas strains, the inventors first selected the, Pseudomonas citronellolis strain and incubated UV-treated PE (UV-PE) with the selected Pseudomonas citronellolis strain. After 30 days, the UV-PE was recovered from the flasks, and analyzed via a high temperature gel permeation chromatography (HT-GPC) polymer analysis. [0197] When compared to a test control (PE only; no strain added), the inventors observed about 25% of a weight averaged molecular weight (Mw) reduction of from the sample incubated with the selected strain and about 33% of a number averaged molecular weight (Mn) reduction (FIGs. 3A-3B). This observation of PE degradation demonstrates that microbes can degrade PE.

EXAMPLE 4: Trametes versicolor produced enzyme leads to a 50% reduction in molecular weight of polyethylene

[0198] To test whether Trametes versicolor laccase (purified powder; Sigma Aldrich 38429) and a reaction mediator (i.e., HBT; hydroxybenzotriazole) can facilitate PE degradation by reduction of molecular weight distribution (MWD), the inventors incubated PE with T. versicolor laccase and HBT (reaction mediator). After 30 days, the PE was recovered and analyzed via a high temperature gel permeation chromatography (HT-GPC) polymer analysis.

[0199] When compared to a test control (PE only; no enzyme added), the inventors observed about 44% of a weight averaged molecular weight (Mw) reduction of from the sample incubated with the laccase and HBT, and about 56% of a number averaged molecular weight (Mn) reduction (FIGs. 4A-4B). This observation of PE degradation demonstrates that laccases can degrade PE. EXAMPLE 5: Trametes versicolor produced enzyme oxidatively degrades polyethylene [0200] T. versicolor laccase (purified powder; Sigma Aldrich 38429) and HBT were incubated with PE for 14 days to see if the laccase- mediator system (LMS) can oxidatively degrade PE. Then, the PE was recovered, and analyzed via a Fourier Transform Infrared Spectroscopy (FTIR) analysis. This analysis was conducted with 5 mL reaction containing 250 mg PE powder, 0.5 mM HBT, 100 mM citrate buffer (5.0), 0.05% Triton X-100, and 1 mg laccase applied 5 times over 14 days.

[0201] From this experiment, the reaction product shows clear signatures of PE oxidation in comparison to the laccase-untreated control, as shown in FIG. 5.

EXAMPLE 6: Enzymatic pretreatment with a Trametes versicolor produced enzyme before exposure to a Pseudomonas strain could lead to a synergistic polyethylene degradation system

[0202] In order to compare PE oxidation effect by two distinct treatments, (i) laccase (Sigma Aldrich 38429) and (ii) UV-irradiation, the inventors set up two conditions; (i) incubation of T. versicolor laccase with PE for 14 days and (ii) UV treatment to PE for 34 days (UV irradiation). Then, the PEs were recovered from each treatment, and analyzed via a Fourier Transform Infrared Spectroscopy (FTIR) analysis.

[0203] The results from the FTIR analyses between laccase treatment and UV treatment indicate that both treatments can give similar PE oxidation effects. In addition to the reduction in MW with T. versicolor laccase (Example 4), the laccase appears to oxidize PE with results similar to that of UV-irradiation (FIG. 6), suggesting that enzymatic pretreatment could replace UV-irradiation. [0204] Thus, the inventors confirm that enzymatic pretreatment with a T. versicolor produced enzyme before exposure to a Pseudomonas strain could lead to a synergistic polyethylene degradation system.

EXAMPLE 7: Pseudomonas strains cultured on UV-irradiated PE increase in biomass [0205] Pseudomonas citronellolis strain was cultured on UV-irradiated PE for 10 days in order to see any increase in biomass, and observed about 5 times increase in biomass relative to the strain cultured on UV-untreated PE, suggesting UV-treated PE is a preferred carbon source (FIGs. 7A- 7B).

EXAMPLE 8: Pseudomonas strains cultured on a variety of PE substrates [0206] To examine how the Pseudomonas citronellolis (P6) strain grows on a variety of PE substrates, nine different PE substrates were prepared to incubate with the P6 strain. P. citronellolis (P6) growth is presented in FIG. 8 on nine different PE substrates. The tested PE substrates are as follows; four untreated PE substrates (PEI to PE4) and five UV-treated PE substrates (UV-PE1 to UV-PE4). UV-PE1 has two treatment conditions, which are UV-PEl-1 and UV-PE1-2, depending on a time period of UV treatment (i) UV-PEl-1 : 30 days UV treatment on PEI; (ii) UV-PE1-2: 72 days UV treatment on PEI). NA indicates a negative control (No PE).

[0207] As seen in FIG. 8, the P6 strains reacted on a variety of PEs with preference to UV- pretreated PE substrates over untreated PE substrates.

EXAMPLE 9: Growth of P. citronellolis (P6) strain on laccase-pretreated and/or UV- pretreated polyethylene (PE)

[0208] To test how P. citronellolis (P6) strain grows on PE pretreated by laccase or UV, experiments were performed as follows. First, a pre-seed culture was started from 50 uL of a glycerol stock of P6 strain in 50 mL LB media, grown shaking at 200 rpm for 24 h at 37°C. This was used to inoculate a 50 mL seed culture at an OD600 of 0.5 in M9 minimal media + 2% glucose, which was incubated shaking at 200 rpm for 16 h at 30°C. After this, P6 cells were washed to remove any residual media components and used to inoculate main cultures at an OD of 0.01 in M9 minimal media, 2 mg/mL PE (untreated, UV-, or laccase-pretreated), and 0.05% Triton X-100 (used to disperse the PE substrates in liquid). Cultures were maintained shaking at 1000 rpm at 30°C for 8 days. The main culture negative control contained all culture components except for PE.

[0209] Since the particulate nature of PE interferes with typical spectrophotometric measurements of microbial growth, an assay was developed in Phase 0 to measure cell growth by measuring DNA concentrations in cultures. This was accomplished by adapting a standard DNA quantification assay using the PicoGreen reagent, which selectively binds double stranded DNA to form a fluorescent complex. At each time point, main cultures were sampled and lysed using CHAPS buffer before freezing at -80°C. At the end of the experiment, all lysed samples were thawed, mixed, and transferred to plates containing the PicoGreen reagent. Additional samples were transferred via a serial dilution to the plate for constructing a standard curve to convert PicoGreen values to OD600. These were prepared with/ ⁵ . citronellolis that were grown in glucose media to a known OD600 (as measured in a spectrophotometer), lysed and frozen identically to the PE culture samples. Fluorescence measurements (Ex: 480 nm / Em: 520 nm) were taken using a Spark instrument (Tecan). For all experiments, negative growth controls containing all culture components except for UV-SPE were sampled and measured with the resulting values used for background subtraction from experimental data.

[0210] FIG. 9 illustrates P. citronellolis growth for 8 days on (i) laccase-pretreated (20 day pretreatment) PE, (ii) untreated PE, and (iii) UV-pretreated (28-day pretreatment) PE. Starting OD600 = 0.01. This result shows that P. citronellolis strain preferred laccase-pretreated PE and UV-pretreated PE substrates, when compared to untreated PE substrate, indicating that pretreatment by laccase or UV can facilitate degradation or bioassimilation of PE substrates by P. citronellolis.

EXAMPLE 10: Growth of P. nitroreducens (P8) strain on laccase-pretreated and/or UV- pretreated polyethylene (PE)

[0211] To test how P. P. nitroreducens (P8) strain grows on PE pretreated by laccase or UV, experiments were performed as follows. First, a pre-seed culture was started from 50 uL of a glycerol stock of P. nitroreducens in 50 mL LB media, grown shaking at 200 rpm for 24 h at 37°C. This was used to inoculate a 50 mL seed culture at an OD600 of 0.5 in M9 minimal media + 2% hexadecane, which was incubated shaking at 200 rpm for 24 h at 30°C. After this, cells were washed to remove any residual media components and used to inoculate main cultures at an OD of 0.01 in SM0 minimal media, 12 mg/mL PE (UV- or laccase-pretreated), and 0.05% Triton X- 100 (used to disperse the PE substrates in liquid). Cultures were maintained shaking at 1000 rpm at 30°C for 7 days. The main culture negative control contained all culture components except for PE.

[0212] The P8 cell growth was measured in the same way as described in Example 9. FIG. 10 illustrates P. nitroreducens growth for 7 days on (i) negative control (No PE), (ii) laccase- pretreated (20 day pretreatment) PE, and (iii) UV-pretreated (28-day pretreatment) PE. Starting OD600 = 0.01. FIG. 10 tells that/ ⁵ . nitroreducens strain preferred laccase-pretreated PE and UV- pretreated PE substrates, when compared to the negative control (without PE) that was not distinguishable from growth on the untreated PE substrate (data not shown). This data indicates that P. nitroreducens can grow faster and thereby more efficiently reduce molecular weight of PE substrates pretreated by laccase or UV.

EXAMPLE 11: Various combinations for creating polyethylene degradation systems [0213] There are various ways to create polyethylene degradation systems using strains and/or enzymes with different treatment conditions such as UV treatment and/or a reaction mediator taught in the previous examples. One way is a two-step fermentation of PE by pretreating PE with laccase and fermenting the pre-treated PE as shown in FIG. 11 A. The laccase-pretreated PE can be used as feedstock for bacterial bioassimilation by the strains that would facilitate fermentation of the laccase-pretreated PE.

[0214] Another way is a single-step fermentation of PE with engineered bioassimilation strain in which laccases are expressed, as shown in FIG. 11B. The genetically-engineered strains that express and produce an enzyme such as laccase can be used for bacterial bioassimilation of PE. Thus, the engineered bioassimilation strains have been generated and are being screened for further testing.

[0215] To make the engineered bioassimilation strains with laccase exogenously expressed, recombinant DNA constructs comprising a nucleic acid sequence encoding a laccase protein have been generated and are being transformed into host cells or strains of interest [0216] The laccase expression and its enzymatic activity will be tested from the newly generated bioassimilation strains. Then, the aforementioned genetically-engineered strains, in which laccase is functionally expressed, will be utilized to ferment and/or degrade PE substrates for bioassimilation and biodegradation of PE.

INCORPORATION BY REFERENCE

[0217] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0218] It should be understood that the above description is only representative of illustrative embodiments and examples. For the convenience of the reader, the above description has focused on a limited number of representative examples of all possible embodiments, examples that teach the principles of the disclosure. The description has not attempted to exhaustively enumerate all possible variations or even combinations of those variations described. That alternate embodiments may not have been presented for a specific portion of the disclosure, or that further undescribed alternate embodiments may be available for a portion, is not to be considered a disclaimer of those alternate embodiments. One of ordinary skill will appreciate that many of those undescribed embodiments, involve differences in technology and materials rather than differences in the application of the principles of the disclosure. Accordingly, the disclosure is not intended to be limited to less than the scope set forth in the following claims and equivalents.

REFERENCES CITED

[0219] Bhatia et al, 2014 “Implications of a novel Pseudomonas species on low density polyethylene biodegradation: an in vitro to in silico approach” SpringerPlus [0220] Montazer et al, 2019 “Microbial degradation of low-density polyethylene and synthesis of polyhydroxyalkanoate polymers” Can. J. Microbiol

[0221] Montazer et al, 2018 “Microbial Degradation of UV-Pretreated Low-Density Polyethylene Films by Novel Polyethylene-Degrading Bacteria Isolated from Plastic Dump Soil” J. Polym Environ

[0222] Kyaw et al, 2012, “Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species” Indian J. Microbiol

[0223] Fujisawa et al, 2002, “Degradation of Polyethylene and Nylon-66 by the Laccase- Mediator System” J Polym Environ 9, 103-108

[0224] Kim C, Lorenz WW, Hoopes JT, Dean JF. Oxidation of phenolate siderophores by the multicopper oxidase encoded by the Escherichia coli yacK gene. J Bacteriol. 2001;183:4866-75 [0225] Morozova OV, Shumakovich GP, Shleev SV, Yaropolov YI. Laccase-mediator systems and their applications: a review. Appl Biochem Microbiol. 2007;43:523-35 [0226] Upadhyay, P., Shrivastava, R. & Agrawal, P.K. Bioprospecting and biotechnological applications of fungal laccase. 3 Biotech 6, 15 (2016).

[0227] Arregui, L., Ayala, M., Gomez-Gil, X. et al. Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact 18, 200 (2019)

[0228] Montazer, Zahra et al. “Challenges with Verifying Microbial Degradation of Polyethylene.” Polymers vol. 12(1) 123 (2020)

NUMBERED EMBODIMENTS OF THE DISCLOSURE

Provided below are various embodiments of the disclosure, which illustrate various aspects of the disclosure.

1. A method of degrading polyethylene, the method comprising: a. combining a Pseudomonas microbe and/or an oxidase enzyme with a polyethylene substrate, wherein the polyethylene substrate’s molecular weight is reduced upon exposure to the

Pseudomonas microbe and/or the oxidase enzyme.

2. The method according to embodiment 1 , wherein the Pseudomonas microbe is present.

3. The method according to embodiment 1 or 2, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens or Pseudomonas citronellolis .

4. The method according to any one of embodiments 1-3, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens.

5. The method according to any one of embodiments 1 -4, wherein the Pseudomonas microbe is present and is Pseudomonas citronellolis strain ATCC 13674 or Pseudomonas citronellolis strain NRRL B-2504 or Pseudomonas citronellolis strain DSM 50332.

6. The method according to any one of embodiments 1 -5, wherein the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe.

7. The method according to any one of embodiments 1-6, wherein the polyethylene substrate comprises a polyethylene selected from a group consisting of a ultra-high-molecular- weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HDPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE).

8. The method according to any one of embodiments 1-7, wherein the polyethylene substrate is a powder or film.

9. The method according to any one of embodiments 1-8, wherein the polyethylene substrate is a powder. The method according to any one of embodiments 1-9, wherein the polyethylene substrate is a low-molecular weight polyethylene powder. The method according to any one of embodiments 1-10, wherein the polyethylene substrate is mixed or commingled with an additive. The method according to any one of embodiments 1-11, wherein the polyethylene substrate is a mixed polyethylene plastic article. The method according to any one of embodiments 1-12, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The method according to any one of embodiments 1-13, wherein the non-ionic surfactant is Triton X-100. The method according to any one of embodiments 1-14, wherein the polyethylene substrate is UV-pretreated. The method according to any one of embodiments 1-15, wherein the polyethylene substrate is oxidase enzyme-pretreated. The method according to any one of embodiments 1-16, wherein the polyethylene substrate is not UV-pretreated before combining with the Pseudomonas microbe and/or the oxidase enzyme. The method according to any one of embodiments 1-17, wherein the oxidase enzyme is present. The method according to any one of embodiments 1-18, wherein the oxidase enzyme is present and comprises a multi-copper center structure. The method according to any one of embodiments 1-19, wherein the oxidase enzyme is present and is a laccase. The method according to any one of embodiments 1-20, wherein the oxidase enzyme is present and is a bacterial derived oxidase enzyme. The method according to any one of embodiments 1-21, wherein the oxidase enzyme is present and is a fungal derived oxidase enzyme. The method according to any one of embodiments 1-22, wherein the oxidase enzyme is present and is a laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. The method according to any one of embodiments 1-23, wherein the laccase is derived from Trametes versicolor strain ATCC 42530, Trametes versicolor strain ATCC 42462, Trametes versicolor strain ATCC 20869, Trametes versicolor strain ATCC 96186,

Trametes versicolor strain ATCC 42394, Trametes versicolor strain ATCC 20547,

Trametes versicolor strain ATCC 11235, Trametes versicolor strain ATCC 200801, Trametes versicolor strain ATCC 48424, Trametes versicolor strain ATCC 34584,

Trametes versicolor strain ATCC 58078, Trametes versicolor strain ATCC 66173,

Trametes versicolor strain DSM 1977, Trametes versicolor strain DSM 3086, Trametes versicolor strain DSM 6401, Trametes versicolor strain DSM 11269, Trametes versicolor strain DSM 11309, or Trametes versicolor strain NRRL 66313. The method according to any one of embodiments 1-24, wherein the oxidase enzyme is present and is utilized to pre-treat the polyethylene, before the Pseudomonas microbe is combined with the polyethylene. The method according to any one of embodiments 1-25, wherein both the Pseudomonas microbe and the oxidase enzyme are present. The method according to any one of embodiments 1-26, wherein the polyethylene substrate’s molecular weight is reduced by at least 10%, 20%, 30%, 40%, or 50%. A method of degrading polyethylene, the method comprising: a. pretreating a polyethylene substrate by UV or an oxidase enzyme; and b. combining a Pseudomonas microbe with the polyethylene substrate pretreated by UV or the oxidase enzyme of step a), wherein the polyethylene substrate’s molecular weight is reduced upon a sequential exposure to (i) UV or the oxidase enzyme and (ii) the Pseudomonas microbe. The method according to embodiment 28, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens or Pseudomonas citronellolis . The method according to embodiment 28 or 29, wherein the oxidase enzyme is present and is a laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. The method according to any one of embodiments 28-30, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The method according to any one of embodiments 28-31, wherein the non-ionic surfactant is Triton X-100. A composition comprising: a Pseudomonas microbe and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV or an oxidase enzyme. The composition of embodiment 33, wherein the oxidase enzyme is a laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. The composition according to embodiment 33 or 34, wherein the laccase is derived from

Trametes versicolor strain ATCC 42530, Trametes versicolor strain ATCC 42462,

Trametes versicolor strain ATCC 20869, Trametes versicolor strain ATCC 96186,

Trametes versicolor strain ATCC 42394, Trametes versicolor strain ATCC 20547,

Trametes versicolor strain ATCC 11235, Trametes versicolor strain ATCC 200801, Trametes versicolor strain ATCC 48424, Trametes versicolor strain ATCC 34584,

Trametes versicolor strain ATCC 58078, Trametes versicolor strain ATCC 66173,

Trametes versicolor strain DSM 1977, Trametes versicolor strain DSM 3086, Trametes versicolor strain DSM 6401, Trametes versicolor strain DSM 11269, Trametes versicolor strain DSM 11309, or Trametes versicolor strain NRRL 66313. The composition according to any one of embodiments 33-35, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens . The composition according to any one of embodiments 33-36, wherein the Pseudomonas microbe is present and is Pseudomonas citronellolis strain ATCC 13674 or Pseudomonas citronellolis strain NRRL B-2504 or Pseudomonas citronellolis strain DSM 50332. The composition according to any one of embodiments 33-37, wherein the, Pseudomonas microbe is present and the polyethylene is assimilated by the microbe. The composition according to any one of embodiments 33-38, wherein the polyethylene substrate is selected from a group consisting of a ultra-high-molecular- weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HDPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE). The composition according to any one of embodiments 33-39, wherein the polyethylene substrate is a powder or film. The composition according to any one of embodiments 33-40, wherein the polyethylene substrate is a powder. The composition according to any one of embodiments 33-41, wherein the polyethylene substrate is a low-molecular weight polyethylene powder. The composition according to any one of embodiments 33-42, wherein the polyethylene substrate is mixed or commingled with an additive. The composition according to any one of embodiments 33-43, wherein the polyethylene substrate is a mixed polyethylene plastic article. The composition according to any one of embodiments 33-44, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The composition according to any one of embodiments 33-45, wherein the non-ionic surfactant is Triton X-100. A composition comprising: a Pseudomonas microbe, an oxidase enzyme, and a polyethylene substrate. The composition of embodiment 47, wherein the oxidase enzyme is a laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. The composition according to embodiment 47 or 48, wherein the laccase is derived from

Trametes versicolor strain ATCC 42530, Trametes versicolor strain ATCC 42462,

Trametes versicolor strain ATCC 20869, Trametes versicolor strain ATCC 96186,

Trametes versicolor strain ATCC 42394, Trametes versicolor strain ATCC 20547,

Trametes versicolor strain ATCC 11235, Trametes versicolor strain ATCC 200801, Trametes versicolor strain ATCC 48424, Trametes versicolor strain ATCC 34584,

Trametes versicolor strain ATCC 58078, Trametes versicolor strain ATCC 66173,

Trametes versicolor strain DSM 1977, Trametes versicolor strain DSM 3086, Trametes versicolor strain DSM 6401, Trametes versicolor strain DSM 11269, Trametes versicolor strain DSM 11309, or Trametes versicolor strain NRRL 66313. The composition according to any one of embodiments 47-49, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens . The composition according to any one of embodiments 47-50, wherein the Pseudomonas microbe is present and is Pseudomonas citronellolis strain ATCC 13674 or Pseudomonas citronellolis strain NRRL B-2504 or Pseudomonas citronellolis strain DSM 50332. The composition according to any one of embodiments 47-51, wherein the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe. The composition according to any one of embodiments 47-52, wherein the polyethylene substrate is selected from a group consisting of a ultra-high-molecular- weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HOPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE). The composition according to any one of embodiments 47-53, wherein the polyethylene substrate is a powder or film. The composition according to any one of embodiments 47-54, wherein the polyethylene substrate is a powder. The composition according to any one of embodiments 47-55, wherein the polyethylene substrate is a low-molecular weight polyethylene powder. The composition according to any one of embodiments 47-56, wherein the polyethylene substrate is mixed or commingled with an additive. The composition according to any one of embodiments 47-57, wherein the polyethylene substrate is a mixed polyethylene plastic article. The composition according to any one of embodiments 47-58, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The composition according to any one of embodiments 47-59, wherein the non-ionic surfactant is Triton X-100. A composition comprising: a Pseudomonas microbe and an effective amount of a purified oxidase enzyme, wherein a polyethylene substrate is treated with the composition. The composition of embodiment 61, wherein the oxidase enzyme is a laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor. The composition according to embodiment 61 or 62, wherein the laccase is derived from

Trametes versicolor strain ATCC 42530, Trametes versicolor strain ATCC 42462,

Trametes versicolor strain ATCC 20869, Trametes versicolor strain ATCC 96186,

Trametes versicolor strain ATCC 42394, Trametes versicolor strain ATCC 20547,

Trametes versicolor strain ATCC 11235, Trametes versicolor strain ATCC 200801, Trametes versicolor strain ATCC 48424, Trametes versicolor strain ATCC 34584, Trametes versicolor strain ATCC 58078, Trametes versicolor strain ATCC 66173, Trametes versicolor strain DSM 1977, Trametes versicolor strain DSM 3086, Trametes versicolor strain DSM 6401, Trametes versicolor strain DSM 11269, Trametes versicolor strain DSM 11309, or Trametes versicolor strain NRRL 66313. The composition according to any one of embodiments 61-63, wherein the Pseudomonas microbe is present and is Pseudomonas nitroreducens . The composition according to any one of embodiments 61-64, wherein the Pseudomonas microbe is present and is Pseudomonas citronellolis strain ATCC 13674 or Pseudomonas citronellolis strain NRRL B-2504 or Pseudomonas citronellolis strain DSM 50332. The composition according to any one of embodiments 61-65, wherein the Pseudomonas microbe is present and the polyethylene is assimilated by the microbe. The composition according to any one of embodiments 61-66, wherein the polyethylene substrate is selected from a group consisting of a ultra-high-molecular- weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HDPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE). The composition according to any one of embodiments 61-67, wherein the polyethylene substrate is a powder or film. The composition according to any one of embodiments 61-68, wherein the polyethylene substrate is a powder. The composition according to any one of embodiments 61-69, wherein the polyethylene substrate is a low-molecular weight polyethylene powder. The composition according to any one of embodiments 61-70, wherein the polyethylene substrate is mixed or commingled with an additive. The composition according to any one of embodiments 61-71, wherein the polyethylene substrate is a mixed polyethylene plastic article. The composition according to any one of embodiments 61-72, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The composition according to any one of embodiments 61-73, wherein the non-ionic surfactant is Triton X-100. A composition comprising: a Pseudomonas microbe, a laccase and a polyethylene substrate, wherein the polyethylene substrate is pretreated by UV. The composition of embodiment 75, wherein the laccase is derived from Trametes versicolor. The composition according to embodiment 75 or 76, wherein the polyethylene substrate is selected from a group consisting of a ultra-high-molecular-weight polyethylene (UHMWPE), a ultra-low-molecular-weight polyethylene (ULMWPE or PE-WAX), a high-molecular-weight polyethylene (HMWPE), a high-density polyethylene (HOPE), a high-density cross-linked polyethylene (HDXLPE), a cross-linked polyethylene (XLPE), a medium-density polyethylene (MDPE), a linear low-density polyethylene (LLDPE), a low-density polyethylene (LDPE), a very-low-density polyethylene (VLDPE), and a chlorinated polyethylene (CPE). The composition according to any one of embodiments 75-77, wherein the polyethylene substrate is a powder or film. The composition according to any one of embodiments 75-78, wherein the polyethylene substrate is a powder. The composition according to any one of embodiments 75-79, wherein the polyethylene substrate is a low-molecular weight polyethylene powder. The composition according to any one of embodiments 75-80, wherein the polyethylene substrate is mixed or commingled with an additive. The composition according to any one of embodiments 75-81, wherein the polyethylene substrate is a mixed polyethylene plastic article. The composition according to any one of embodiments 75-82, wherein the polyethylene substrate is exposed to an non-ionic surfactant. The composition according to any one of embodiments 75-83, wherein the non-ionic surfactant is Triton X-100. A microbial strain capable of degrading PE, comprising: a. P. citronellolis ATCC 13674 = NRRL B-2504 = DSM 50332 or b. P. nitroreducens DSM 14399 enzyme (e.g. laccase) capable of degrading PE, comprising: a. T. versicolor laccase E substrate dispersed in aqueous medium using a surfactant, comprising: a. low molecular weight PE powder; b. other low molecular weight PE powders to be growth substrates for the microbes but not amenable to enzymatic degradation by the laccase; and c. the surfactant used is Triton X-l 00 ymatic assay conditions including a mediator of laccase activity, comprising: a. mediator - Hydroxybenzotriazole (HBT) ethod to detect growth in PE-containing cultures, comprising: a. preparing sample using a cell lysis method, with DNA detection using picogreen DNA dye ethod for quantitative recovery of PE from cultures or enzyme assays, comprising: a. using bleach to dissolve biomaterial (microbial cultures) or SDS to remove enzyme (enzyme assays) b. performing filtration and quantitative recovery of PE powder to detect any mass loss ethod for analyzing molecular weight of recovered PE, comprising: a. performing molecular weight analysis