Activators for utilization of pet, derivatives and / or monomers thereof as the carbon source

Abstract

A recombinant actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, wherein the actinobacterium in its native / wildtype or unactivated state cannot metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source. Also provided are uses of streptomyces antibiotic regulatory protein (SARP) regulators to activate or enhance metabolism of PET, a derivative thereof and / or a monomer thereof as a carbon source in an actinobacterium; a method of degrading PET, a derivative thereof and / or a monomer thereof; and a method of determining if an actinobacterium can metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

Description

[0001]

[0002] ACTIVATORS FOR UTILIZATION OF PET, DERIVATIVES AND / OR MONOMERS THEREOF AS THE CARBON SOURCE

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to a recombinant actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, wherein the actinobacterium in its native / wildtype or unactivated state cannot metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

[0005] The disclosure also relates to uses of Streptomyces antibiotic regulatory protein (SARP) regulators to activate or enhance metabolism of PET, a derivative thereof and / or a monomer thereof as a carbon source in an actinobacterium; a method of degrading PET, a derivative thereof and / or a monomer thereof; and a method of determining if an actinobacterium can metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

[0006] BACKGROUND

[0007] The biological degradation of PET (polyethylene terephthalate) plastic is a process in which microorganisms break down the polymer chains of PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). The discovery of polyethylene terephthalate hydrolase (PETase) from Ideonella sakaiensis in 2016 has opened the possibility of engineering biological hosts for PET degradation. The two monomers, TPA and EG, have dramatically different solubilities in aqueous systems. TPA may be easily recovered from the aqueous environment, where microorganisms are typically incubated, due to its low solubility in water. EG, on the other hand, is fully miscible in water, making isolation difficult. Thus, PET labelled as “biologically recycled PET” can at most be 50% recycled from biological sources.

[0008] One major disadvantage of biological recycling is cost. Biological systems need to be constantly fed with food and nutrients to maintain consistent performance. EG, which accumulates in the system as PET breaks down, may be considered as a carbon source for the system, thereby reducing costs of the associated process. At the same time, this will also reduce EG toxicity levels in the system. More importantly, with the availability of EG that typically require 1) extensive and costly purification / downstream processing to recover, and 2) cannot be upcycled easily, identifying ways to activate EG metabolism in strains that would enable such EG to be easily recycled (as carbon source for PET enzymes producing strains), or upcycled (to other higher value products) are highly attractive.

[0009] Certain microorganisms such as Pseudomonas putida have been shown to metabolize EG naturally as a carbon source. Esc / ?enc / ? / a co / / , which does not naturally metabolise EG, have been successfully engineered with heterologous enzymatic pathways to enable them to utilise EG as a carbon source.

[0010] Nonetheless, there is a need for a general strategy for enabling microorganisms which do not naturally metabolise PET or its derivatives to metabolise such materials.

[0011] SUMMARY

[0012] In one aspect, there is provided a recombinant actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising a transgene encoding a Streptomyces Antibiotic Regulatory Protein (SARP) regulator, wherein the actinobacterium in its native / wildtype or unactivated state cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

[0013] In one aspect, there is provided a use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to activate metabolism of polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof in an actinobacterium, which in its native / wildtype or unactivated state cannot metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

[0014] In one aspect, there is provided a use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to enhance metabolism of polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof in an actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

[0015] In one aspect, there is provided a use of a recombinant actinobacterium according to any preceding claim for degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof.

[0016] In one aspect, there is provided a method of degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof comprising contacting the polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof with the recombinant actinobacterium according to any preceding claim.

[0017] In one aspect, there is provided a method of determining if an actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising the step of:

[0018] • culturing the actinobacterium in a media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof, wherein the media does not comprise any alternative carbon source; and one or more of the following steps:

[0019] • checking if bacteria colonies can form;

[0020] • microscopic analysis of the PET, derivative thereof and / or monomer thereof before and after culturing the actinobacterium;

[0021] • measuring the change in weight of the PET, derivative thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis.

[0022] • determining if there is an increase in biomass production, for example by optical density (OD) measurements; and

[0023] • determining if there is an increase in the formation of PET monomers.

[0024] In one embodiment, the method further comprises the steps of:

[0025] • culturing the actinobacterium in a media comprising a positive control carbon source that the actinobacterium known to be able to metabolise; and one or more of the following steps:

[0026] • checking if bacteria colonies can form

[0027] • microscopic analysis of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium;

[0028] • measuring the change in weight of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis; and

[0029] • determining if there is an increase in biomass production, for example by optical density (OD) measurements.

[0030] In one embodiment: if bacteria colonies can form in both the media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof and the media comprising the positive control carbon source, then the actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source; or if bacteria colonies only form in the media comprising the positive control carbon source, then the actinobacterium cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source;

[0031] • if there is a significant degradation in the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;

[0032] • if there is a significant decrease in weight of the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;

[0033] • if there is a significant increase in biomass production after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source; or

[0034] • if there is a significant increase in the formation of PET monomers after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source.

[0035] In one embodiment, the checking step for ethylene glycol (EG) or terephthalic acid (TPA) metabolism comprises visually checking for bacterial colonies or comprises harvesting a bacterial pellet and visualising the pellet under a microscope.

[0036] In one embodiment, the media is a minimal media.

[0037] In one embodiment, the minimal media comprises one or more of the following:

[0038] • K2HPO4 (dipotassium phosphate)

[0039] • MgSC (magnesium sulfate)

[0040] • FeSC Oron (II) sulfate); and

[0041] • (NH4)2SC>4 (ammonium sulfate)

[0042] In one embodiment, the minimal media is around neutral pH.

[0043] In one embodiment, the media comprises up to 25% EG.

[0044] In one embodiment, the media comprises 5% EG.

[0045] In one embodiment, the media comprises 5 mg / ml or more of PET.

[0046] In one embodiment, the media comprises 0.5 to 1.5 mM TPA.

[0047] In one embodiment, the positive control carbon source is a sugar.

[0048] In one embodiment, the sugar is 0.5 to 2% glucose.

[0049] In one embodiment, the actinobacterium is cultured for up to 14 days before checking if bacteria colonies can form.

[0050] In one embodiment, the actinobacterium which cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source is identified by employing the method as described above.

[0051] In one embodiment, the PET derivative thereof is selected from the group comprising a PET microplastic, a PET nanoplastic, a PET oligomer and a PET copolymer.

[0052] In one embodiment, the PET monomer is selected from the group comprising ethylene glycol (EG), terephthalic acid (TPA), dimethyl terephthalate (DMT), mono-2- hydroxyethyl terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET).

[0053] In one embodiment, the PET monomer is selected from the group comprising EG, TPA and DMT.

[0054] In one embodiment, the actinobacterium in its native / wildtype or unactivated state cannot metabolise one or more of the following as a carbon source: PET, EG, TPA, DMT, BHET and / or MHET.

[0055] In one embodiment, the actinobacterium cannot metabolise:

[0056] • EG as a carbon source,

[0057] • PET as a carbon source,

[0058] • EG and PET as a carbon source,

[0059] • PET and TPA as a carbon source,

[0060] • EG and TPA as a carbon source, or

[0061] • EG, PET and TPA as a carbon source.

[0062] In one embodiment, the actinobacterium is selected from the group comprising: streptomyces, actinomyces, corynebacterium, nocardiopsis, Arthrobacter, solwaraspora, Frankia, micromonospora, micrococcus, nocardia, plantactinospora, salinispora and saccharomonospora. Amycolatopsis and Microbispora.

[0063] In one embodiment, the actinobacterium is a strain of Streptomyces.

[0064] In one embodiment, the Streptomyces is selected from the group comprising: Streptomyces fradiae, Streptomyces albogriseolus, Streptomyces albofaciens, Streptomyces albus, Streptomyces abietis, Streptomyces brasiliscabiei, Streptomyces hayashii, Streptomyces alfalfa, Streptomyces andamanensis, Streptomyces alkaliphilus, Streptomyces albiflavescens, Streptomyces acidicola, Streptomyces avermitilis, Streptomyces osmaniensis, Streptomyces actuosus, Streptomyces antibioticus, Streptomyces misionensis, Streptomyces aquilus, Streptomyces angustmyceticus, Streptomyces atratus, Streptomyces coelicolor, Streptomyces griseus, Streptomyces amakusaensis, Streptomyces acidiscabies, Streptomyces albicerus, Streptomyces adustus, Streptomyces asenjonii, Streptomyces altiplanensis, Streptomyces albaduncus, Streptomyces spongiicola, Streptomyces achromogenes, Streptomyces abikoensis, Streptomyces naganishii, Streptomyces racemochromogenes, Streptomyces albidoflavus, Streptomyces aurantiacus, Streptomyces auratus, Streptomyces althioticus, Streptomyces boili, Streptomyces hilarionis, Streptomyces actinomycinicus, Streptomyces arcticus, Streptomyces aridus, Streptomyces abyssomicinicus, Streptomyces alkaliterrae, Streptomyces chiangmaiensis, Streptomyces monomycini, Streptomyces amritsarensis, Streptomyces thermocarboxydovorans, Streptomyces kaempferi, Streptomyces tacrolimicus, Streptomyces ambofaciens, Streptomyces anulatus and Streptomyces aureoverticillatus. Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces coelicoflavus, Streptomyces wuyuanensis, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces rimosus, and Streptomyces sp. CB02923.

[0065] In one embodiment, the actinobacterium is selected from the group comprising: Streptomyces fradiae, Streptomyces albogriseolus and Streptomyces albofaciens.

[0066] In one embodiment, the actinobacterium is a strain of Streptomyces selected from the group comprising A61715, T265, T298 and T343.

[0067] In one embodiment, the SARP regulator activates / enhances metabolism of one or more of the following in the actinobacterium: PET, EG, TPA, DMT, BHET and / or MHET.

[0068] In one embodiment, the SARP regulator activates / enhances:

[0069] • EG metabolism in the actinobacterium,

[0070] • PET metabolism in the actinobacterium,

[0071] • TPA metabolism in the actinobacterium,

[0072] • EG and PET metabolism in the actinobacterium,

[0073] • PET and TPA metabolism in the actinobacterium,

[0074] • EG and TPA metabolism in the actinobacterium, or

[0075] • EG, PET and TPA metabolism in the actinobacterium.

[0076] In one embodiment, the SARP regulator has a BTAD (bacterial transcriptional activation domain) from SARP.

[0077] In one embodiment, the SARP regulator comprises or consists of a BTAD and an OBD (OmpR DNA binding domain).

[0078] In one embodiment, the SARP regulator is a full length SARP regulator.

[0079] In one embodiment, the SARP regulator is selected from the group comprising: RedD, S5 SARP, S6 SARP, AdpA, FAS, Crp, SarA, polY, RslR3, Orf4, FilR, PimR, PteR, ScnRI, PnR2, PoIR, SanG, BafG, CpkO, BenR, FdmR1, SnoA, SCAB1371 , CdaR, Atr32, ArpRI, AsuR5, ChlF2, CpkN, MilR3 / KelR, MonRI, NanR1 , NanR2, NigR, VmsR, VmsS, TylS, Actll-Orf4, Alb45, AlpV, Aur1 PR3, Aur1 PR4, Dnrl, MilR3 / KelR, MtmR, OtcR, RslR1 , RslR2, SrcmRI, Txn9, PieR, BulY, BulZ, CcaR, PapR1 , PapR2, PapR4, SrrY, SrrZ, FarR3, FarR4, ThnU, SgvR2, SgvR3, Orf22, Vlml, NosP, and PlaR1.

[0080] In one embodiment, the SARP regulator is selected from the group comprising RedD and S5 SARP.

[0081] In one embodiment, the SARP regulator is RedD SARP having an amino acid sequence as set forth in SEQ ID NO: 1 or S5 SARP having an amino acid sequence as set forth in SEQ ID NO: 2.

[0082] In one embodiment, the SARP regulator is RedD and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces chrestomyceticus, Streptomyces monomycini, Streptomyces sp. CB02923, Streptomyces rimosus, Streptomyces albofaciens, Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, and Streptomyces wuyuanensis.

[0083] In one embodiment, the SARP regulator is RedD and the actinobacterium is:

[0084] • Streptomyces fradiae, wherein RedD activates EG, PET and / or TPA metabolism in Streptomyces fradiae;

[0085] • Streptomyces albogriseolus, wherein RedD activates PET metabolism in Streptomyces albogriseolus;

[0086] • Streptomyces spongiicola, wherein RedD activates PET metabolism in Streptomyces spongiicola;

[0087] • Streptomyces tirandamycinicus wherein RedD activates PET metabolism in Streptomyces tirandamycinicus;

[0088] • Streptomyces sp. CWH03, wherein RedD activates PET metabolism in Streptomyces sp. CWH03;

[0089] • Streptomyces coelicoflavus, wherein RedD activates PET metabolism in Streptomyces coelicoflavus;

[0090] • Streptomyces wuyuanensis, wherein RedD activates PET metabolism in Streptomyces wuyuanensis;

[0091] • Streptomyces thermolilacinus, wherein RedD activates PET metabolism in Streptomyces thermolilacinus;

[0092] • Streptomyces roseolilacinus, wherein RedD activates PET metabolism in Streptomyces roseolilacinus;

[0093] • Streptomyces viridodiastaticus, wherein RedD activates PET metabolism in Streptomyces viridodiastaticus;

[0094] • Streptomyces werraensis, wherein RedD activates PET metabolism in Streptomyces werraensis;

[0095] • Streptomyces thermocarboxydus, wherein RedD activates PET metabolism in Streptomyces thermocarboxydus;

[0096] • Streptomyces gancidicus, wherein RedD activates PET metabolism in Streptomyces gancidicus', or

[0097] • Streptomyces tendae, wherein RedD activates PET metabolism in Streptomyces tendae.

[0098] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae. Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces wuyuanensis.

[0099] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is:

[0100] • Streptomyces fradiae, wherein S5 SARP activates EG and PET metabolism in Streptomyces fradiae;

[0101] • Streptomyces albogriseolus, wherein S5 SARP activates EG metabolism in Streptomyces albogriseolus;

[0102] • Streptomyces albofaciens, wherein S5 SARP activates PET, EG and / or TPA metabolism in Streptomyces albofaciens;

[0103] • Streptomyces chrestomyceticus, wherein S5 SARP activates PET metabolism in Streptomyces chrestomyceticus;

[0104] • Streptomyces monomycini, wherein S5 SARP activates PET metabolism in Streptomyces monomycini;

[0105] • Streptomyces sp.CB02923, wherein S5 SARP activates PET metabolism in Streptomyces sp. CB02923;

[0106] • Streptomyces rimosus, wherein S5 SARP activates PET metabolism in Streptomyces rimosus;

[0107] • Streptomyces spongiicola, wherein S5 SARP activates PET metabolism in Streptomyces spongiicola;

[0108] • Streptomyces tirandamycinicus, wherein S5 SARP activates PET metabolism in Streptomyces tirandamycinicus;

[0109] • Streptomyces sp. CWH03, wherein S5 SARP activates PET metabolism in Streptomyces sp. CWH03;

[0110] • Streptomyces coelicoflavus, wherein S5 SARP activates PET metabolism in Streptomyces coelicoflavus;

[0111] • Streptomyces wuyuanensis, wherein S5 SARP activates PET metabolism in Streptomyces wuyuanensis;

[0112] • Streptomyces thermolilacinus, wherein S5 SARP activates EG metabolism in Streptomyces thermolilacinus;

[0113] • Streptomyces roseolilacinus, wherein S5 SARP activates EG metabolism in Streptomyces roseolilacinus',

[0114] • Streptomyces_viridodiastaticus, wherein S5 SARP activates Eg metabolism in Streptomyces_viridodiastaticus;

[0115] • Streptomyces werraensis, wherein S5 SARP activates EG metabolism in Streptomyces werraensis;

[0116] • Streptomyces thermocarboxydus, wherein S5 SARP activates EG metabolism in Streptomyces thermocarboxydus;

[0117] • Streptomyces gancidicus, wherein S5 SARP activates EG metabolism in Streptomyces gancidicus; or

[0118] • Streptomyces tendae, wherein S5 SARP activates EG metabolism in Streptomyces tendae.

[0119] DEFINITIONS

[0120] As used herein the term “actinobacterium” refers to a diverse group of Grampositive bacteria characterized by a high, typically greater than 55 mol%, guanine and cytosine (G + C) content in their DNA. These bacteria can be aerobic or facultative and are known for their rod-shaped or mycelium-like filamentous structures.

[0121] Actinobacteria belong to the order Actinomycetales and are notable for their ability to form spores. They are ubiquitous in nature, found in various habitats including soil, water, and as part of the normal flora in the digestive tracts of animals. They play a crucial role in decomposing organic matter, thereby contributing to soil health and nutrient cycling.

[0122] Actinobacteria includes several classes such as:

[0123] • Actinomycetia: Members of this class include many medically and economically significant bacteria, such as those in the order Actinomycetales. Streptomyces, which is a genus within this class, is particularly notable for producing antibiotics.

[0124] • Acidimicrobiia: Members of this class are known fortheir ability to thrive in acidic environments. They play a role in the decomposition of organic matter and nutrient cycling.

[0125] • Nitriliruptoria: Members of this class are involved in the degradation of nitriles, which are organic compounds containing a cyano group. They play a role in nitrogen cycling.

[0126] • Rubrobacteria, which are known for their red pigmentation and ability to survive in extreme environments, such as high radiation and desiccation.

[0127] • Coriobacteriia: This class includes bacteria that are part of the human gut microbiome and help maintain the mucosal barrier and reduce lipopolysaccharide in the intestine.

[0128] • Thermoleophilia, which are adapted to high-temperature environments. They contribute to the decomposition of organic matter in hot springs and other geothermal habitats .

[0129] The terms “native” and “wildtype” are used interchangeably herein to refer to the non-mutated, non-recombinant version of an actinobacterium that is typically found in nature.

[0130] As used herein, an actinobacterium in its ‘unactivated state’ has not yet undergone the activation process to enable the actinobacterium to metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source. Thus, an actinobacterium in its unactivated state could be a recombinant actinobacterium (i.e. not a wildtype / native actinobacterium) that is unable to metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

[0131] As used herein the term “Streptomyces Antibiotic Regulatory Protein (SARP) regulator” refers to a family of transcription regulators that are found primarily in actinomycetes, particularly Streptomyces. SARP regulators are characterised by a conserved SARP domain, which comprises an N-terminal OmpR-type DNA binding domain (ODB) and a C-terminal bacterial transcriptional activation domain (BTAD). In addition to an N-terminal SARP domain, larger SARP regulators may comprise additional domains such as a central NB-ARC domain and a C-terminal tetratricopeptide repeat (TPR) domain.

[0132] SARP regulators are pathway-specific activators of secondary metabolite biosynthesis and have been shown to be highly efficient in upregulation of both global and specific production of secondary metabolites. They are associated with various antibiotic gene clusters, including ribosomal and non-ribosomal synthesized peptides, type I and type ll-PKS derived polyketides, hybrid polyketide-peptide compounds, and azoxy compounds. SARP genes are typically located within the biosynthetic gene cluster that they regulate. Prior to the present disclosure, no SARP regulator has been known to be associated with enabling non-glucose based feedstock utilisation.

[0133] Within the context of the present disclosure ‘a derivative of PET’ refers to any derivative of PET wherein PET is used as the starting material to produce the derivative. Examples of PET derivatives include, but are not limited to, PET microplastics, PET nanoplastics, PET oligomers, and co-polymers of PET. Examples of PET co-polymers include but are not limited to:

[0134] • PET-co-2,7-naphthalate, which is PET that has been co-polymerised with 2,7-naphthalate and exhibits improved thermal, mechanical and barrier properties;

[0135] • PET-co-MPDiol, which is PET that has been co-polymerised with MPDiol glycol;

[0136] • PET-co-isophthalic acid, which is PET that has been co-polymerised with purified isophthalic acid (PIA), improving its processability and properties;

[0137] • PET-co-polycaprolactone, which is a co-polymer of PET and polycaprolactone (PCL); and

[0138] • PET-co-PE, which is a co-polymer of PET and polyethylene (PE).

[0139] As used herein ‘a monomer of PET’ refers to any monomer that reacts with other monomers to form the PET polymer. Examples of PET monomers include ethylene glycol (EG), terephthalic acid (TPA), dimethyl terephthalate (DMT), mono-2-hydroxyethyl terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET).

[0140] As used herein the term “media” refers to any liquid or solution which provides essential nutrients, growth factors and / or environmental conditions required to support actinobacteria growth in vitro. Examples of commonly used media for actinobacteria culture include but are not limited to ISP-2 medium, Kenknight medium, starch casein medium, humic acid vitamin agar, GYM (glucose / yeast extract / malt extract), etc.

[0141] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0142] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

[0143] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1 .01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0144] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0145] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0146] DESCRIPTION OF EMBODIMENTS

[0147] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

[0148] Streptomyces Antibiotic Regulatory Protein (SARP)-type regulators, found primarily in actinomycetes, particularly Streptomyces, are pathway-specific activators of secondary metabolite biosynthesis. These have been shown to be highly efficient in upregulation of both global and specific production of secondary metabolites. They are associated with various antibiotic gene clusters, including type I and type ll-PKS derived polyketides, ribosomal and non-ribosomal synthesized peptides, hybrid polyketide- peptide compounds, p-azachinones, and azoxy compounds. SARP genes are typically located within the biosynthetic gene cluster that they regulate. However, currently no known SARP gene is associated with enabling non-glucose based feedstock utilisation.

[0149] Unexpectedly, the present inventors have demonstrated that SARP-type regulators are able to activate utilisation of PET and its derivatives and hydrolysed monomers, ethylene glycol, in actinobacteria, particularly in Streptomyces strains such as Streptomyces fradiae and Streptomyces albogriseolus.

[0150] Thus, in one aspect, there is provided the use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to activate metabolism of polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof, in an actinobacterium which cannot metabolise PET in its native / wildtype or unactivated state, a derivative thereof and / or a monomer thereof as a carbon source.

[0151] Advantageously, the presently disclosed use enables an actinobacterium which normally cannot metabolise PET, a derivative thereof and / or a monomer thereof, to use one or more of these as a carbon source. This provides a versatile and efficient tool for genome engineering and optimization, especially in facilitating the effective utilization of next-generation non-food biomass feedstock.

[0152] Hence, in another aspect, there is provided a recombinant actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising a transgene encoding a Streptomyces Antibiotic Regulatory Protein (SARP) regulator, wherein the wild type actinobacterium cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

[0153] In yet another aspect, there is provided the use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to enhance polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof metabolism in an actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

[0154] Without being bound by theory, the present inventors believe that SARP regulators may also be able to enhance the existing ability of an actinobacterium to metabolise PET, a derivative thereof and / or a monomer thereof.

[0155] In one aspect, there is provided the use of a recombinant actinobacterium as described above for degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof.

[0156] In one aspect, there is provided a method of degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof comprising contacting the polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof with the recombinant actinobacterium according to any preceding AS.

[0157] In yet another aspect, there is provided a method of determining if an actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising the step of:

[0158] • culturing the actinobacterium in a media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof, wherein the media does not comprise any alternative carbon source; and one or more of the following steps: o checking if bacteria colonies can form; o microscopic analysis of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium; o measuring the change in weight of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis; o determining if there is an increase in biomass production, for example by optical density (OD) measurements; and o determining if there is an increase in the formation of PET monomers. Advantageously, the disclosed method provides a variety of different ways by which the skilled addressee can assess whether or not an actinobacterium can metabolise PET, a derivative thereof and / or a monomer thereof. This may be helpful for assessing if any one of the above disclosed uses has been successfully implemented; for example, to check for successful activation of metabolism of PET, a derivative thereof and / or a monomer thereof in an actinobacterium that previously was unable to metabolise any of the above.

[0159] In one embodiment, the method further comprises:

[0160] • culturing the actinobacterium in a media comprising a positive control carbon source that the actinobacterium known to be able to metabolise; and one or more of the following steps: o checking if bacteria colonies can form o microscopic analysis of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium; o measuring the change in weight of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis; and o determining if there is an increase in biomass production, for example by optical density (OD) measurements.

[0161] In one embodiment of the method:

[0162] • if bacteria colonies can form in both the media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof and the media comprising the positive control carbon source, then the actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source; or if bacteria colonies only form in the media comprising the positive control carbon source, then the actinobacterium cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source;

[0163] • if there is a significant degradation in the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;

[0164] • if there is a significant decrease in weight of the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;

[0165] • if there is a significant increase in biomass production after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source; or

[0166] • if there is a significant increase in the formation of PET monomers after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source.

[0167] In one embodiment, the checking step for ethylene glycol (EG) or terephthalic acid (TPA) metabolism comprises visually checking for bacterial colonies.

[0168] In one embodiment, the checking step for PET metabolism comprises harvesting a bacterial pellet and visualizing the pellet under a microscope.

[0169] In one embodiment, the media is a minimal media.

[0170] In one embodiment, the minimal media comprises one or more of the following:

[0171] • K2HPO4 (dipotassium phosphate)

[0172] • MgSC (magnesium sulfate)

[0173] • FeSC (iron (II) sulfate); and

[0174] • (NH4)2SO4 (ammonium sulfate)

[0175] In one embodiment, the minimal media is around neutral pH, for example 7.0 to 7.2, such as 7.0, 7.1 or 7.2.

[0176] In one embodiment, the media comprises up to 25% EG, for example 5%, 10%, 15%, 20% or 25% EG, in particular 5% EG.

[0177] In one embodiment, the media comprises 15 to 25% EG, such as 18 to 22% EG, for example 18, 19, 20, 21 or 22% EG, in particular 20% EG.

[0178] In one embodiment, the media comprises 5 mg / mL or more of PET, such as 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL.

[0179] In one embodiment, the media comprises 0.5 to 1.5 mM TPA, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, or 1.50 mM TPA.

[0180] In one embodiment, the positive control carbon source is a sugar, for example glucose, sucrose or galactose.

[0181] In one embodiment, the sugar is glucose, for example 0.5 to 2% glucose, such as 0.5, 1.0, 1.5 or 2% glucose, in particular 1% glucose.

[0182] In one embodiment, the actinobacterium is cultured for up to 14 days before checking if bacteria colonies can form, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, such as 4 days, in particular 14 days.

[0183] In one embodiment, the actinobacterium which cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source is identified by employing the method as described herein.

[0184] In one embodiment, the PET monomer is selected from the group comprising ethylene glycol (EG), terephthalic acid (TPA), dimethyl terephthalate (DMT), mono-2- hydroxyethyl terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET).

[0185] In one embodiment, the PET monomer is selected from the group comprising ethylene glycol (EG), terephthalic acid (TPA) and dimethyl terephthalate (DMT).

[0186] In one embodiment, the PET monomer is selected from the group comprising ethylene glycol (EG) and terephthalic acid (TPA).

[0187] In one embodiment, the PET monomer is EG.

[0188] In one embodiment, the PET monomer is TPA.

[0189] In one embodiment, the PET monomer is DMT.

[0190] In one embodiment, the PET monomer is MHET.

[0191] In one embodiment, the PET monomer is BHET.

[0192] In one embodiment, the actinobacterium in its native / wildtype or unactivated state cannot metabolise one or more of the following as a carbon source: PET, EG, TPA, DMT, BHET and / or MHET.

[0193] In one embodiment, the actinobacterium cannot metabolise EG as a carbon source.

[0194] In one embodiment, the actinobacterium cannot metabolise PET as a carbon source.

[0195] In one embodiment, the actinobacterium cannot metabolise TPA as a carbon source.

[0196] In one embodiment, the actinobacterium cannot metabolise BHET as a carbon source.

[0197] In one embodiment, the actinobacterium cannot metabolise MHET as a carbon source.

[0198] In one embodiment, the actinobacterium cannot metabolise EG and PET as a carbon source.

[0199] In one embodiment, the actinobacterium cannot metabolise PET and TPA as a carbon source.

[0200] In one embodiment, the actinobacterium cannot metabolise EG and TPA as a carbon source.

[0201] In one embodiment, the actinobacterium cannot metabolise EG, PET and TPA as a carbon source.

[0202] In one embodiment, the actinobacterium is selected from the group comprising:

[0203] Streptomyces, Actinomyces, Corynebacterium, Nocardiopsis, Arthrobacter, Solwaraspora, Frankia, Micromonospora, Micrococcus, Nocardia, Plantactinospora, Salinispora and Saccharomonospora, Amycolatopsis, Microbispora.

[0204] In one embodiment, the actinobacterium is a strain of Streptomyces.

[0205] In one embodiment, the Streptomyces is selected from the group comprising: Streptomyces fradiae, Streptomyces albogriseolus, Streptomyces albofaciens,

[0206] Streptomyces albus, Streptomyces abietis, Streptomyces brasiliscabiei, Streptomyces hayashii, Streptomyces alfalfa, Streptomyces andamanensis, Streptomyces alkaliphilus, Streptomyces albiflavescens, Streptomyces acidicola, Streptomyces avermitilis, Streptomyces osmaniensis, Streptomyces actuosus, Streptomyces antibioticus, Streptomyces misionensis, Streptomyces aquilus, Streptomyces angustmyceticus, Streptomyces atratus, Streptomyces coelicolor, Streptomyces griseus, Streptomyces amakusaensis, Streptomyces acidiscabies, Streptomyces albicerus, Streptomyces adustus, Streptomyces asenjonii, Streptomyces altiplanensis, Streptomyces albaduncus, Streptomyces spongiicola, Streptomyces achromogenes, Streptomyces abikoensis, Streptomyces naganishii, Streptomyces racemochromogenes, Streptomyces albidoflavus, Streptomyces aurantiacus, Streptomyces auratus, Streptomyces althioticus, Streptomyces boili, Streptomyces hilarionis, Streptomyces actinomycinicus, Streptomyces arcticus, Streptomyces aridus, Streptomyces abyssomicinicus, Streptomyces alkaliterrae, Streptomyces chiangmaiensis, Streptomyces monomycini, Streptomyces amritsarensis, Streptomyces thermocarboxydovorans, Streptomyces kaempferi, Streptomyces tacrolimicus, Streptomyces ambofaciens, Streptomyces anulatus and Streptomyces aureoverticillatus, Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces coelicoflavus, Streptomyces wuyuanensis, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces rimosus, and Streptomyces sp. CB02923.

[0207] In one embodiment, the Streptomyces is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae. Streptomyces chrestomyceticus, Streptomyces monomycini, Streptomyces sp. CB02923, Streptomyces rimosus, Streptomyces albofaciens, Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces wuyuanensis.

[0208] In one embodiment, the actinobacterium is Streptomyces fradiae. In one embodiment, the actinobacterium is Streptomyces albogriseolus. In one embodiment, the actinobacterium is Streptomyces albofaciens.

[0209] In one embodiment, the actinobacterium is a strain of Streptomyces selected from the group comprising A61715, T265, T298 and T343.

[0210] In one embodiment, the actinobacterium is Streptomyces strain A61715.

[0211] In one embodiment, the actinobacterium is Streptomyces strain T265.

[0212] In one embodiment, the actinobacterium is Streptomyces strain T298.

[0213] In one embodiment, the actinobacterium is Streptomyces strain T343.

[0214] In one embodiment, the SARP regulator activates / enhances metabolism of one or more of the following in the actinobacterium: PET, EG, TPA, DMT, BHET and / or MHET, for example EG, PET, TPA, EG and PET, PET and TPA, EG and TPA or EG, PET and TPA metabolism.

[0215] In one embodiment, the SARP regulator activates / enhances EG metabolism in the actinobacterium.

[0216] In one embodiment, the SARP regulator activates / enhances PET metabolism in the actinobacterium.

[0217] In one embodiment, the SARP regulator activates / enhances TPA metabolism in the actinobacterium.

[0218] In one embodiment, the SARP regulator activates / enhances DMT metabolism in the actinobacterium.

[0219] In one embodiment, the SARP regulator activates / enhances BHET metabolism in the actinobacterium.

[0220] In one embodiment, the SARP regulator activates / enhances MHET metabolism in the actinobacterium.

[0221] In one embodiment, the SARP regulator activates / enhances EG and PET metabolism in the actinobacterium.

[0222] In one embodiment, the SARP regulator activates / enhances PET and TPA metabolism in the actinobacterium.

[0223] In one embodiment, the SARP regulator activates / enhances EG and TPA metabolism in the actinobacterium.

[0224] In one embodiment, the SARP regulator activates / enhances EG, PET and TPA metabolism in the actinobacterium.

[0225] In one embodiment, the SARP regulator is a full length SARP regulator, for example the SARP regulator comprises the full length sequence of RedD SARP or the full length sequence of S5 SARP.

[0226] In one embodiment, the SARP regulator is truncated, i.e. does not comprise the full length sequence of a SARP regulator.

[0227] In one embodiment, the SARP regulator has a BTAD (bacterial transcriptional activation domain) from SARP, for example a fusion protein comprising a BTAD.

[0228] In one embodiment, the SARP regulator comprises a BTAD and an OBD (OmpR DNA binding domain) from a SARP regulator. Without being bound by theory, the present inventors believe that incorporating only the BTAD and OBD of a SARP regulator may be sufficient to activate / enhance metabolism of PET, its derivatives thereof and / or monomers thereof. Thus, in one embodiment, the SARP regulator consists of a BTAD and an OBD. In one embodiment, the SARP regulator lacks an NTPase domain. In one embodiment, the SARP regulator lacks a TPR (tetratricopeptide repeat) domain.

[0229] In one embodiment, the SARP regulator is selected from the group comprising: RedD, S5 SARP, S6 SARP, AdpA, FAS, Crp, SarA, polY, RslR3, Orf4, FilR, PimR, PteR, ScnRI, PnR2, PoIR, SanG, BafG, CpkO, BenR, FdmR1, SnoA, SCAB1371, CdaR, Atr32, ArpRI, AsuR5, ChlF2, CpkN, MilR3 / KelR, MonRI, NanR1 , NanR2, NigR, VmsR, VmsS, TylS, Actll-Orf4, Alb45, AlpV, Aur1 PR3, Aur1 PR4, Dnrl, MIIR3 / KelR, MtmR, OtcR, RslR1 , RslR2, SrcmRI, Txn9, PieR, BulY, BulZ, CcaR, PapR1 , PapR2, PapR4, SrrY, SrrZ, FarR3, FarR4, ThnU, SgvR2, SgvR3, Orf22, Vlml, NosP, and PlaR1.

[0230] In one embodiment, the SARP regulator is selected from the group comprising RedD and S5 SARP.

[0231] In one embodiment, the SARP regulator is selected from the group comprising RedD and S5 SARP.

[0232] In one embodiment, the SARP regulator is RedD, for example having the amino acid sequence as set forth in SEQ ID NO: 1.

[0233] In one embodiment, the SARP regulator is S5 SARP, for example having the amino acid sequence as set forth in SEQ ID NO: 2.

[0234] Thus, in one embodiment, the SARP regulator has an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0235] In one embodiment, the SARP regulator has an amino acid sequence as set forth in SEQ ID NO: 1.

[0236] In one embodiment, the SARP regulator has an amino acid sequence as set forth in SEQ ID NO: 2.

[0237] In one embodiment, the SARP regulator is RedD and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus,

[0238] Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces chrestomyceticus, Streptomyces monomycini, Streptomyces sp. CB02923, Streptomyces rimosus, Streptomyces albofaciens, Streptomyces spongiicola,

[0239] Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces wuyuanensis.

[0240] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces fradiae.

[0241] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces fradiae; and wherein RedD activates EG, PET and / or TPA metabolism in Streptomyces fradiae.

[0242] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces albogriseolus.

[0243] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces albogriseolus; and wherein RedD activates PET metabolism in Streptomyces albogriseolus.

[0244] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces spongiicola; for example, wherein RedD activates PET metabolism in Streptomyces spongiicola.

[0245] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces tirandamycinicus; for example, wherein RedD activates PET metabolism in Streptomyces tirandamycinicus.

[0246] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces sp. CWH03; for example, wherein RedD activates PET metabolism in Streptomyces sp. CWH03.

[0247] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces coelicoflavus; for example, wherein RedD activates PET metabolism in Streptomyces coelicoflavus.

[0248] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces wuyuanensis; for example, wherein RedD activates PET metabolism in Streptomyces wuyuanensis.

[0249] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces thermolilacinus; for example, wherein RedD activates PET metabolism in Streptomyces thermolilacinus.

[0250] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces roseolilacinus; for example, wherein RedD activates PET metabolism in Streptomyces roseolilacinus.

[0251] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces viridodiastaticus; for example, wherein RedD activates PET metabolism in Streptomyces viridodiastaticus.

[0252] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces werraensis; for example, wherein RedD activates PET metabolism in Streptomyces werraensis.

[0253] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces thermocarboxydus'. for example, wherein RedD activates PET metabolism in Streptomyces thermocarboxydus.

[0254] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces gancidicus; for example, wherein RedD activates PET metabolism in Streptomyces gancidicus.

[0255] In one embodiment, the SARP regulator is RedD and the actinobacterium is Streptomyces tendae; for example, wherein RedD activates PET metabolism in Streptomyces tendae.

[0256] In one embodiment, the SARP regulator is RedD and the actinobacterium is a Streptomyces strain selected from the group comprising A61715, T265, T298 and T343; for example, wherein RedD activates PET, EG and / or TPA metabolism in one or more of these strains.

[0257] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae. Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces wuyuanensis.

[0258] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces fradiae.

[0259] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces fradiae; and w1herein S5 SARP activates EG and PET metabolism in Streptomyces fradiae.

[0260] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces albogriseolus.

[0261] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces albogriseolus; and wherein S5 SARP activates EG metabolism in Streptomyces albogriseolus.

[0262] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces albofaciens.

[0263] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces albofaciens; and wherein S5 SARP activates PET, EG and / or TPA metabolism in Streptomyces albofaciens.

[0264] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces chrestomyceticus; for example, wherein S5 SARP activates PET metabolism in Streptomyces chrestomyceticus.

[0265] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces monomycini; for example, wherein S5 SARP activates PET metabolism in Streptomyces monomycini.

[0266] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces sp. CB02923; for example, wherein S5 SARP activates PET metabolism in Streptomyces sp. CB02923.

[0267] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces rimosus; for example, wherein S5 SARP activates PET metabolism in Streptomyces rimosus.

[0268] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces spongiicola; for example, wherein S5 SARP activates PET metabolism in Streptomyces spongiicola.

[0269] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces tirandamycinicus; for example, wherein S5 SARP activates PET metabolism in Streptomyces tirandamycinicus.

[0270] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces sp. CWH03; for example, wherein S5 SARP activates PET metabolism in Streptomyces sp. CWH03.

[0271] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces coelicoflavus; for example, wherein S5 SARP activates PET metabolism in Streptomyces coelicoflavus.

[0272] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces wuyuanensis, for example, wherein S5 SARP activates PET metabolism in Streptomyces wuyuanensis.

[0273] The use, actinobacterium or method according to any preceding AS, wherein the SARP regulator is S5 SARP and the actinobacterium is Streptomyces thermolilacinus, for example, wherein S5 SARP activates EG metabolism in Streptomyces thermolilacinus.

[0274] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces roseolilacinus; for example, wherein S5 SARP activates EG metabolism in Streptomyces roseolilacinus.

[0275] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces viridodiastaticus; for example, wherein S5 SARP activates EG metabolism in Streptomyces viridodiastaticus.

[0276] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces werraensis; for example, wherein S5 SARP activates EG metabolism in Streptomyces werraensis.

[0277] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces thermocarboxydus; for example, wherein S5 SARP activates EG metabolism in Streptomyces thermocarboxydus.

[0278] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces gancidicus; for example, wherein S5 SARP activates EG metabolism in Streptomyces gancidicus.

[0279] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is Streptomyces tendae; for example, wherein S5 SARP activates EG metabolism in Streptomyces tendae.

[0280] In one embodiment, the SARP regulator is S5 SARP and the actinobacterium is a Streptomyces strain selected from the group comprising T265, T298 and T343; for example, wherein SARP activates PET, EG and / or TPA metabolism in one or more of these strains.

[0281] In one aspect, there is provided a use substantially as described herein, for example with reference to the drawings.

[0282] In one aspect, there is provided an actinobacterium substantially as described herein, for example with reference to the drawings.

[0283] In one aspect, there is provided a method substantially as described herein, for example with reference to the drawings.

[0284] BRIEF DESCRIPTION OF FIGURES

[0285] Figure 1 shows a schematic diagram of domains within the RedD and S5 SARP regulators. OBD: OmpR DNA binding domain, BTAD: bacterial activation domain, NTPase: nucleoside triphosphate hydrolases domain.

[0286] Figure 2 shows graphs comparing the growth of strains on minimal media with and without PET. (A) T265 mutants with PET - endpoint ODeoo(t = 15 days). (B) A61715 m5b mutant with PET - endpoint ODeoo (t = 15 days). (C) T298 m3b mutant with PET - endpoint ODeoo (t = 15 days). (D) T343 m4w2 mutant with PET - endpoint ODeoo (t = 15 days). Measurements of media alone with and without PET is also shown as negative control (Media). The experiments were conducted in triplicates.

[0287] Figure 3 shows a graph comparing the growth of strains on minimal media with and without 5% EG. T298 and T343 mutants with 5% EG - endpoint ODeoo (t = 15 days). Measurements of media alone with and without EG is also shown as negative control (no cells). All experiments were conducted in triplicates. Only T298 649-2’s no EG measurements were taken in duplicates.

[0288] Figure 4 shows a representative 20x microscope image of PET resuspended in minimal media. If the morphology of a sample (bacteria strain + PET) resembles Figure 4 with the presence of large intact granules, this indicates that PET granules are not broken down or utilised by the microbes.

[0289] Figure 5 shows the results of the T265 m3b mutant with integrated overexpression cassette for the RedD SARP regulator grown in minimal media with various carbon sources. Photo of samples and representative 20x microscope image of pellet sample after 14 days when grown in minimal media with PET or glucose as a carbon source.

[0290] Figure 6 shows the results of the T265 m4a mutant with integrated overexpression cassette for the S5 SARP regulator grown in minimal media with various carbon sources. Photo of samples and representative 20x microscope image of pellet sample after 14 days when grown in minimal media with PET or glucose as a carbon source.

[0291] Figure 7 shows a photo of samples, and representative 20x microscope image of pellet sample the T298 m3b mutant with integrated overexpression cassette for the RedD SARP regulator grown in minimal media with PET or glucose as a carbon source after 14 days.

[0292] Figure 8 shows the results of the T298 m4b mutant with integrated overexpression cassette for the S5 SARP regulator grown in minimal media with various carbon sources. Photo of samples after 4 days when grown in minimal media with EG or glucose as a carbon source.

[0293] Figure 9 shows a photo of samples and representative 20x microscope image of pellet sample of T343 m4w2 mutant with integrated overexpression cassette for the RedD SARP regulator grown in minimal media with PET or glucose as a carbon source after 14 days.

[0294] Figure 10 shows a photo of samples and representative 20x microscope image of pellet sample of the A61715 m5b mutant with integrated overexpression cassette for the S5 SARP regulator grown in minimal media with PET or glucose as a carbon source after 14 days.

[0295] Figure 11 shows graphs indicating the quantities of TPA (A), MHET (B), and BHET (C) produced from PET metabolism by A61715 and its m5b mutant (S5 SARP). Detection was conducted using UHPLC-MS on a 150 pL sample of cultivation media that was collected after 15 days. The control sample corresponds to the media without cells. Another control was established to assess the baseline levels of monomers related to the same cell and media combination, omitting PET (media). Experiments were conducted in triplicates.

[0296] Figure 12 shows graphs indicating the quantity of TPA (A), MHET (B), or BHET (C) generated from PET metabolism by T265 and its mutants — specifically, T265 m4a (which features the integrated S5 SARP overexpression cassette) and T265 m3b (which includes the integrated RedD). Detection was conducted using UHPLC-MS on a 150 pL sample of cultivation media that was collected after 15 days. The control sample corresponds to the media without cells (control). Another control was established to assess the baseline levels of monomers related to the same cell and media combination, omitting PET (media). Experiments were conducted in triplicates.

[0297] Figure 13 shows graphs indicating the amount of TPA (A), MHET (B), or BHET (C) produced from PET metabolism by T298 and its m3b mutant (RedD). Detection was conducted using UHPLC-MS on a 150 L sample of cultivation media that was collected after 15 days. The control sample corresponds to the media without cells. Another control was established to assess the baseline levels of monomers related to the same cell and media combination, omitting PET (media). Experiments were conducted in triplicates.

[0298] EXAMPLES

[0299] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0300] Example 1 - Proof of concept experiments

[0301] Bacterial strains used in study

[0302] The bacterial strains used in the experiments are shown in Table 1 below.

[0303] Table 1 - Description of strains used in this study. PhyloPhlAn, an integrated pipeline for large-scale phylogenetic profiling of genomes and metagenomes, is utilized to compute the distance between new strains and others, thereby characterizing their genomic similarity. This distance value provides insights into the phylogeny of the strains.

[0304] SARP regulators integrated into bacterial strains

[0305] The SARP regulators are shown in Table 2 below:

[0306] Table 2. Regulators

[0307] The sequences of the regulators are:

[0308] RedD SARP (SEQ ID NO: 1)

[0309] MTGGGVLATMDPVRKLVRSQPKIGRHPVAAGQDGRDRHPIRSWECGERARTARTG

[0310] RTVGRAADPSDHGPSLYNFGGCVEINILGPVSIDTSHSGGGIRAGKVRTLVATLAIDAG

[0311] RAVSLADLVDELWGATPPDNVLNALQAHAARARKVLNERACPERAGGILRSVLGGYL

[0312] LEIDPQCVDGNRFLRLVSQGAALLPADPTRAVELLETGLRLWRGPALIDAGEGRRCR

[0313] GAAALFEERRLTALEDLISAMFLRGGEAQAIAMLQQLVAQYPLRERFCELLMVGLYRV

[0314] GRQGDALESYRLARKRLDDELGVQPGALLRRRHAEILAQDPVLKVPSALWREPYAPA DTSLLSA

[0315] S5 SARP (SEQ ID NO: 2)

[0316] MLDDLQFDVFGPLTVRRNGELLEIPRAKHRVVLAALLLHAPRALTAEELIQQLW GGEPPLTARKTLQGYIARLRKLLGAEAWSRASGYAIAIGAERLDLDRFNTFLHKAELT TEPAERARLFRSAMEQTNGTPLVDIPSEYLQQGDGAALLERWLNATESWADAEMAV GRHAEVLPRLRALVSEHPFRESAWGRLMTALYRAGRQGEALAAYQEARRLLADELG VEPGEELRAAHGRILAGPARSTAQPAPPRSAPAGPRPGAAGAPARRGCPYTLPPGLA EFARPGIEADLIRRLRDVSREGAERTAAQTLNLYGAAGVGKTTMAVRVAHAVSPAFPD GQLYVELQRGDAFREPADVLGELLRALGVGPGAVPADSGERVALYRGLLSHRRVLIV LDGARDEAQVRPLLPASPTCAA VTSLEMLSTLGGTRHVRLGLLTAEESREILARILGR ERVAAEEGAARDLVRYCGRLPLAIRIVGARLLERPHWRLETLARRLESERRRLDELAV GDLGVRDSLAVGYAALDAPERRAFRLLSLLETASFPVRVAAAALGLPDDRTEETLERL TAQHLLEADFVKGSGVRFSYHPLVRLYARELTFDVDPMRSRHGAVQNALAAWYARP AGALVPLRATPSASGPAARAEAVAAAGPLPPELGFTA

[0317] The overexpression cassette for RedD and S5 SARP genes were integrated into the bacterial strains via PhiC31 recombinase as described in Tay et al., 2024.

[0318] In these overexpression cassettes, genes were placed under a strong constitutive promoter, kas*O promoter.

[0319] Wildtype unmodified strains were included as controls.

[0320] Photos and microscope images of samples

[0321] A minimal media was prepared containing potassium phosphate, magnesium sulfate, iron (II) sulfate and ammonium sulfate.

[0322] Next, minimal media (2 mL) containing EG (400 pL), PET (40 mg) or TPA (0.75 nM) were seeded with 150 pL bacteria cells that were previously grown in rich media. Cells were also grown in minimal media supplemented with glucose as a positive control.

[0323] The cells were incubated at 30 °C and 250 rpm for 14 to 20 days. At Days 4 and 14, the samples were checked to determine if the bacteria strains were able to metabolise PET, EG or TPA in the following fashion:

[0324] • For EG orTPA: samples were visually checked and compared with a positive control grown in minimal media containing glucose.

[0325] • For PET: the solid pellet, containing cells and PET were harvested. A sample of the pellet was visualized using a microscope (20x magnification). Figure 4 shows a representative image of what to expect for a sample where the bacteria strain is unable to metabolise PET as a carbon source.

[0326] Growth measurements

[0327] A starter culture was grown overnight using rich media (SV2). 50 L starter culture was seeded into 2-mL minimal media (using a 14-mL round bottom culture tube) with PET (40 mg) or 5% EG as a carbon source. Corresponding controls not containing PET or EG were also grown. Each sample was grown in triplicate. Cultures were incubated at 30 °C with shaking (200 rpm) for 15 days. Thereafter, cell density (ODeoo) was measured by taking a 150-pL aliquot.

[0328] Components of SV2 media (1-L)

[0329] • 15 g glucose

[0330] • 15 g glycerol

[0331] • 15 g soya peptone

[0332] • 1 g CaCOs

[0333] Components of minimal media (1-L)

[0334] • 0.5

[0335] • 0.2

[0336] • 5 g

[0337] • Adjusted to pH 7

[0338] Results

[0339] Graphs summarising the growth measurement results are shown in Figures 2 and 3. Representative photos and microscope images of the samples taken at Days 4 or Day 14 are shown in Figures 5 to 11. The results indicate that:

[0340] T265 m4a (with S5 SARP overexpression cassette integrated) and T265 m3b (RedD integrated) showed significant improvement in growth on PET as sole carbon source, over WT T265. See Figure 2A.

[0341] A61715 m5b (S5 SARP) had significant improvement in growth on PET as sole carbon source over WT A61715. See Figure 2B.

[0342] T298 m3b (RedD) had significant improvement in growth on PET as sole carbon source over WT T298. See Figure 2C.

[0343] T343 m4w2 (RedD) had a significant improvement in growth on PET as the sole carbon source over WT T343. See Figure 2D.

[0344] T343 649-4 (Flag tagged- S5 SARP), T343 650-1 (HA tagged- S5 SARP) had significant improvement in growth on EG as sole carbon source over WT T343. WT T343 have minimal growth in both media. See Figure 3.

[0345] T298 m3b (RedD) showed no significant improvement in growth on EG as sole carbon source over WT T298. However, T298 m4b (S5 SARP) and T298 649-2 (Flag tagged-S5 SARP) had significant growth compared to WT T298 and media without EG. See Figure 3.

[0346] Discussion

[0347] In a general study with Streptomyces, consisting of wild type strains along with integrated mutants (T ay et al, 2024), the present inventors first screened for fast growers which do not form biofilm in the presence of EG. After this, the inventors screened wild type and mutants with SARP regulators integrated in Streptomyces sp. A61715, T343, T298, T265 (See Table 1, Table 2 and Figure 1). The regulators are RedD from Streptomyces coelicolor and SARP regulator from Streptomyces sp. NRRL S-244 (labelled as S5).

[0348] RedD and S5 regulators were not previously or predicted to be associated with ethylene glycol, or PET metabolism. Within SARP family, medium length SARPs (Figure 1) have also been less studied and there are also less of fundamental understanding of these specific regulators. RedD and S5 are found within biosynthetic gene clusters in their respective strains.

[0349] Surprisingly, the inventors found that depending on the strains, integration of RedD and S5 overexpression cassettes were able to activate the utilization of PET in minimal media for biomass production. (See Figure 2). In addition, integration of a S5 overexpression cassette was also able to activate the utilisation of ethylene glycol towards biomass production. (See Figure 3).

[0350] Hence, this Example provides clear evidence of the ability of SARP regulators such as S5 and RedD to activate metabolism of PET and its monomers in bacterial strains that are normally unable to metabolise these materials as a carbon source.

[0351] Example 2 - Follow-up study to investigate metabolism of TPA, MHET and BHET Method

[0352] The strains previously tested for PET metabolism were evaluated for the production of terephthalic acid (TPA), mono-2-hydroxyethyl terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET). An aliquot of the media (150 pL) was mixed with 150 pL of a 0.5 mM solution of 4-bromobenzoic acid in a buffer / methanol mixture (1 :1 , vol / vol). The 4-bromobenzoic acid acted as an internal standard for quantification. The reaction mixture was sonicated, filtered through a 0.2 pm syringe filter, and analysed using UHPLC-MS. The control referenced in Figures 11-12 pertains to the corresponding media without cells. Additionally, another control involves establishing a baseline of monomers in relation to the same cell and media combination, excluding PET.

[0353] Results

[0354] PET polymer is believed to break down into its monomers: terephthalic acid (TPA), or oligomers like mono-2-hydroxyethyl terephthalate (MHET), and bis(2- hydroxyethyl) terephthalate (BHET). Figures 11-13 shows the amounts of TPA, MHET, and BHET produced during fermentations of A61715, T265, T298 and their respective mutants. In both control media and wild-type (WT) strains, similar amounts of TPA, MHET and BHET can be detected, indicating small amounts of background depolymerization under the conditions. However, the present inventors observed only significant increases in TPA levels in the mutants with integrated SARP regulators. This indicates that only the mutants are able to significantly depolymerize PET to its monomers.

[0355] In summary, this study indicates that the integration of SARP regulators has altered the metabolic profile of the actinobacteria, enabling enhanced utilization of PET and its monomers.

[0356] REFERENCES

[0357] Tay, Dillon WP, et al. "Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes." Communications Biology 7.1 (2024): 50.

[0358] APPLICATIONS

[0359] The presently disclosed actinobacterium, uses and methods have various applications, such as the following:

[0360] • Providing versatile and efficient tools for genome engineering and optimisation of actinobacteria, in particular:

[0361] • Use of a Streptomyces Antibiotic Regulatory Protein (SARP) to activate metabolism of PET, its derivatives and / or its monomers in Actinobacterial family, including Streptomyces strains, which do not metabolize ethylene glycol as a carbon source;

[0362] • Use of SARP regulators, including RedD, to activate the metabolism of PET and / or its monomers in A61715, T265, T298, T298 and T343, with genomic similarities to Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae. Streptomyces chrestomyceticus, Streptomyces monomycini, Streptomyces sp_CB02923, Streptomyces rimosus, Streptomyces albofaciens, Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp_CWH03, or Streptomyces wuyuanensis; and

[0363] • Use of medium length SARPs, including S5, to activate the metabolism of PET and / or its monomers in T298 and T343, with genomic similarities to Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomyces_viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, or Streptomyces tendae.

[0364] • Improving carbon source utilisation of next generation non-food feedstock; and

[0365] • Biological recycling of PET, its derivatives therefor and / or its monomers thereof.

[0366] • Treatment or valorization of single or mixed waste streams that may contain PET, its derivatives therefor and / or its monomers thereof.

Claims

1. CLAIMS1 . A recombinant actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising a transgene encoding a Streptomyces Antibiotic Regulatory Protein (SARP) regulator, wherein the actinobacterium in its native / wildtype or unactivated state cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

2. Use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to activate metabolism of polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof in an actinobacterium, which in its native / wildtype or unactivated state cannot metabolise PET, a derivative thereof and / or a monomer thereof as a carbon source.

3. Use of a Streptomyces Antibiotic Regulatory Protein (SARP) regulator to enhance metabolism of polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof in an actinobacterium which can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source.

4. Use of a recombinant actinobacterium according to any preceding claim for degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof.

5. A method of degrading polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof comprising contacting the polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof with the recombinant actinobacterium according to any preceding claim.

6. A method of determining if an actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source, comprising the step of:• culturing the actinobacterium in a media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof, wherein the media does not comprise any alternative carbon source; and one or more of the following steps: o checking if bacteria colonies can form; o microscopic analysis of the PET, derivative thereof and / or monomer thereof before and after culturing the actinobacterium; o measuring the change in weight of the PET, derivative thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis.o determining if there is an increase in biomass production, for example by optical density (OD) measurements; and o determining if there is an increase in the formation of PET monomers.

7. The method according to any one of claims 5 or 6, further comprising the steps of:• culturing the actinobacterium in a media comprising a positive control carbon source that the actinobacterium known to be able to metabolise; and one or more of the following steps: o checking if bacteria colonies can form o microscopic analysis of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium; o measuring the change in weight of the PET, derivate thereof and / or monomer thereof before and after culturing the actinobacterium, for example by HPLC analysis; and o determining if there is an increase in biomass production, for example by optical density (OD) measurements.

8. The method according to any one of claims 5 to 7, wherein:• if bacteria colonies can form in both the media comprising polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof and the media comprising the positive control carbon source, then the actinobacterium can metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source; or if bacteria colonies only form in the media comprising the positive control carbon source, then the actinobacterium cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source;• if there is a significant degradation in the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;• if there is a significant decrease in weight of the PET, the derivative thereof and / or the monomer thereof after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source;• if there is a significant increase in biomass production after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source; or• if there is a significant increase in the formation of PET monomers after culturing the actinobacterium compared to before culturing the actinobacterium, then the actinobacterium can metabolise PET, the derivative thereof and / or the monomer thereof as a carbon source.

9. The method according to any one of claims 5 to 8, wherein the checking step for ethylene glycol (EG) or terephthalic acid (TPA) metabolism comprises visually checking for bacterial colonies or comprises harvesting a bacterial pellet and visualising the pellet under a microscope.

10. The method according to any one of claims 5 to 9, wherein the media is a minimal media.

11. The method according to any one of claims 5 to 10, wherein the minimal media comprises one or more of the following:• K2HPO4 (dipotassium phosphate)• MgSO4 (magnesium sulfate)• FeSC Oron (II) sulfate); and• (NH4)2SO4 (ammonium sulfate)12. The method according to any one of claims 5 to 11 , wherein the minimal media is around neutral pH.

13. The method according to any one of claims 5 to 12, wherein the minimal media is pH 7.0 to 7.2.

14. The method according to any one of claims 5 to 13, wherein the media comprises up to 25% EG.

15. The method according to claim 14, wherein the media comprises 5% EG.

16. The method according to any preceding one of claims 5 to 15, wherein the media comprises 5 mg / ml or more of PET.

17. The method according to any one of claims 5 to 16, wherein the media comprises 0.5 to 1.5 mM TPA.

18. The method according to any one of claims 5 to 17, wherein the positive control carbon source is a sugar.

19. The method according to claim 18, wherein the sugar is 0.5 to 2% glucose.

20. The method according to any one of claims 5 to 19, wherein the actinobacterium is cultured for up to 14 days before checking if bacteria colonies can form.

21. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium which cannot metabolise polyethylene terephthalate (PET), a derivative thereof and / or a monomer thereof as a carbon source is identified by employing the method according to any one of claims 6 to 20 .

22. The actinobacterium, use or method according to any preceding claim, wherein the PET derivative thereof is selected from the group comprising a PET microplastic, a PET nanoplastic, a PET oligomer and a PET co-polymer.

23. The actinobacterium, use or method according to any preceding claim, wherein the PET monomer is selected from the group comprising ethylene glycol (EG), terephthalic acid (TPA), dimethyl terephthalate (DMT), mono-2-hydroxyethyl terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET).

24. The actinobacterium, use or method according to any preceding claim, wherein the PET monomer is selected from the group comprising EG, TPA and DMT.

25. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium in its native / wildtype or unactivated state cannot metabolise one or more of the following as a carbon source: PET, EG, TPA, DMT, BHET and / or MHET.

26. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium cannot metabolise:• EG as a carbon source,• PET as a carbon source,• EG and PET as a carbon source,• PET and TPA as a carbon source,• EG and TPA as a carbon source, or• EG, PET and TPA as a carbon source.

27. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium is selected from the group comprising: streptomyces, actinomyces, corynebacterium, nocardiopsis, Arthrobacter, solwaraspora, Frankia, micromonospora, micrococcus, nocardia, plantactinospora, salinispora and saccharomonospora. Amycolatopsis and Microbispora.

28. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium is a strain of Streptomyces.

29. The actinobacterium, use or method according to claim 28, wherein theStreptomyces is selected from the group comprising: Streptomyces fradiae, Streptomyces albogriseolus, Streptomyces albofaciens, Streptomyces albus, Streptomyces abietis, Streptomyces brasiliscabiei, Streptomyces hayashii, Streptomyces alfalfa, Streptomyces andamanensis, Streptomyces alkaliphilus, Streptomyces albiflavescens, Streptomyces acidicola, Streptomyces avermitilis, Streptomyces osmaniensis, Streptomyces actuosus, Streptomyces antibioticus, Streptomyces misionensis, Streptomyces aquilus, Streptomyces angustmyceticus, Streptomyces atratus, Streptomyces coelicolor, Streptomyces griseus, Streptomyces amakusaensis, Streptomyces acidiscabies, Streptomyces albicerus, Streptomyces adustus, Streptomyces asenjonii, Streptomyces altiplanensis, Streptomyces albaduncus, Streptomyces spongiicola,Streptomyces achromogenes, Streptomyces abikoensis, Streptomyces naganishil, Streptomyces racemochromogenes, Streptomyces albidoflavus, Streptomyces aurantlacus, Streptomyces auratus, Streptomyces althioticus, Streptomyces boili, Streptomyces hilarionis, Streptomyces actinomycinicus, Streptomyces arcticus, Streptomyces aridus, Streptomyces abyssomicinicus, Streptomyces alkaliterrae, Streptomyces chiangmaiensis, Streptomyces monomycini, Streptomyces amritsarensis, Streptomyces thermocarboxydovorans, Streptomyces kaempferi, Streptomyces tacrolimicus, Streptomyces ambofaciens, Streptomyces anulatus and Streptomyces aureoverticillatus. Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces coelicoflavus, Streptomyces wuyuanensis, Streptomyces thermolilacinus, Streptomyces roseolilacinus,Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces rimosus, and Streptomyces sp. CB02923.

30. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium is selected from the group comprising: Streptomyces fradiae, Streptomyces albogriseolus and Streptomyces albofaciens.

31. The actinobacterium, use or method according to any preceding claim, wherein the actinobacterium is a strain of Streptomyces selected from the group comprising A61715, T265, T298 and T343.

32. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator activates / enhances metabolism of one or more of the following in the actinobacterium: PET, EG, TPA, DMT, BHET and / or MHET.

33. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator activates / enhances:• EG metabolism in the actinobacterium,• PET metabolism in the actinobacterium,• TPA metabolism in the actinobacterium,• EG and PET metabolism in the actinobacterium,• PET and TPA metabolism in the actinobacterium,• EG and TPA metabolism in the actinobacterium, or• EG, PET and TPA metabolism in the actinobacterium.

34. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator has a BTAD (bacterial transcriptional activation domain) from SARP.

35. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator comprises or consists of a BTAD and an OBD (OmpR DNA binding domain).

36. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is a full length SARP regulator.

37. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is selected from the group comprising: RedD, S5 SARP, S6 SARP, AdpA, FAS, Crp, SarA, polY, RslR3, Orf4, FilR, PimR, PteR, ScnRI, PnR2, PoIR, SanG, BafG, CpkO, BenR, FdmR1 , SnoA, SCAB1371 , CdaR, Atr32, ArpRI, AsuR5, ChlF2, CpkN, MilR3 / KelR, MonRI, NanR1 , NanR2, NigR, VmsR, VmsS, TylS, Actll-Orf4, Alb45, AlpV, Aur1 PR3, Aur1 PR4, Dnrl, MilR3 / KelR, MtmR, OtcR, RslR1 , RslR2, SrcmRI, Txn9, PieR, BulY, BulZ, CcaR, PapR1 , PapR2, PapR4, SrrY, SrrZ, FarR3, FarR4, ThnU, SgvR2, SgvR3, Orf22, Vim I, NosP, and PlaR1.

38. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is selected from the group comprising RedD and S5 SARP.

39. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is RedD SARP having an amino acid sequence as set forth in SEQ ID NO: 1 or S5 SARP having an amino acid sequence as set forth in SEQ ID NO: 2.

40. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is RedD and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomycesroseolilacinus, Streptomyces coelicoflavus, Streptomyces viridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae, Streptomyces chrestomyceticus, Streptomyces monomycini, Streptomyces sp. CB02923, Streptomyces rimosus, Streptomyces albofaciens, Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, and Streptomyces wuyuanensis.

41. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is RedD and the actinobacterium is:• Streptomyces fradiae, wherein RedD activates EG, PET and / or TPA metabolism in Streptomyces fradiae;• Streptomyces albogriseolus, wherein RedD activates PET metabolism in Streptomyces albogriseolus;• Streptomyces spongiicola, wherein RedD activates PET metabolism in Streptomyces spongiicola;• Streptomyces tirandamycinicus wherein RedD activates PET metabolism in Streptomyces tirandamycinicus;• Streptomyces sp. CWH03, wherein RedD activates PET metabolism in Streptomyces sp. CWH03;• Streptomyces coelicoflavus, wherein RedD activates PET metabolism in Streptomyces coelicoflavus;• Streptomyces wuyuanensis, wherein RedD activates PET metabolism in Streptomyces wuyuanensis;• Streptomyces thermolilacinus, wherein RedD activates PET metabolism in Streptomyces thermolilacinus;• Streptomyces roseolilacinus, wherein RedD activates PET metabolism in Streptomyces roseolilacinus;• Streptomyces viridodiastaticus, wherein RedD activates PET metabolism in Streptomyces viridodiastaticus;• Streptomyces werraensis, wherein RedD activates PET metabolism in Streptomyces werraensis;• Streptomyces thermocarboxydus, wherein RedD activates PET metabolism in Streptomyces thermocarboxydus;• Streptomyces gancidicus, wherein RedD activates PET metabolism in Streptomyces gancidicus; or• Streptomyces tendae, wherein RedD activates PET metabolism in Streptomyces tendae.

42. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is S5 SARP and the actinobacterium is selected from the group comprising Streptomyces fradiae, Streptomyces thermolilacinus, Streptomyces roseolilacinus, Streptomyces coelicoflavus, Streptomycesviridodiastaticus, Streptomyces werraensis, Streptomyces thermocarboxydus, Streptomyces gancidicus, Streptomyces tendae. Streptomyces spongiicola, Streptomyces tirandamycinicus, Streptomyces sp. CWH03, Streptomyces wuyuanensis.

43. The actinobacterium, use or method according to any preceding claim, wherein the SARP regulator is S5 SARP and the actinobacterium is:• Streptomyces fradiae, wherein S5 SARP activates EG and PET metabolism in Streptomyces fradiae',• Streptomyces albogriseolus, wherein S5 SARP activates EG metabolism in Streptomyces albogriseolus',• Streptomyces albofaciens, wherein S5 SARP activates PET, EG and / or TPA metabolism in Streptomyces albofaciens',• Streptomyces chrestomyceticus, wherein S5 SARP activates PET metabolism in Streptomyces chrestomyceticus;• Streptomyces monomycini, wherein S5 SARP activates PET metabolism in Streptomyces monomycini',• Streptomyces sp.CB02923, wherein S5 SARP activates PET metabolism in Streptomyces sp. CB02923;• Streptomyces rimosus, wherein S5 SARP activates PET metabolism in Streptomyces rimosus;• Streptomyces spongiicola, wherein S5 SARP activates PET metabolism in Streptomyces spongiicola;• Streptomyces tirandamycinicus, wherein S5 SARP activates PET metabolism in Streptomyces tirandamycinicus;• Streptomyces sp. CWH03, wherein S5 SARP activates PET metabolism in Streptomyces sp. CWH03;• Streptomyces coelicoflavus, wherein S5 SARP activates PET metabolism in Streptomyces coelicoflavus;• Streptomyces wuyuanensis, wherein S5 SARP activates PET metabolism in Streptomyces wuyuanensis;• Streptomyces thermolilacinus, wherein S5 SARP activates EG metabolism in Streptomyces thermolilacinus;• Streptomyces roseolilacinus, wherein S5 SARP activates EG metabolism in Streptomyces roseolilacinus;• Streptomyces_viridodiastaticus, wherein S5 SARP activates Eg metabolism in Streptomyces_viridodiastaticus;• Streptomyces werraensis, wherein S5 SARP activates EG metabolism in Streptomyces werraensis;• Streptomyces thermocarboxydus, wherein S5 SARP activates EG metabolism in Streptomyces thermocarboxydus;• Streptomyces gancidicus, wherein S5 SARP activates EG metabolism in Streptomyces gancidicus', or• Streptomyces tendae, wherein S5 SARP activates EG metabolism in Streptomyces tendae.

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