Biorecycling of polyester into PHA

Abstract

The present invention provides a method for producing polyhydroxyalkanoates (PHAs) from polyester waste, the method comprising the steps of: (a) providing a culture broth containing polyester waste; and (b) culturing a microorganism in the culture broth to produce PHAs.

Description

[Technical Field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoates (PHAs) from polyester waste. The present invention also relates to the PHAs produced by said method and to articles made using said PHAs.

[0002] [Background technology] Plastics are widely used due to their adaptability, light weight, durability, and flexibility. However, none of the commonly used plastics are biodegradable. As a result, commonly used plastics do not decompose and accumulate in landfills or the natural environment. As of 2015, approximately 6,300 Mt of plastic waste was generated, of which only around 9% was recycled, 12% was incinerated, and 79% accumulated in landfills or the natural environment (see Geyer, R., et al., 2017. Science Advances, 3(7), p. e1700782).

[0003] The degradation of plastics generally occurs slowly in nature and involves various environmental factors such as temperature, moisture, pressure, and microbial activity. To speed up the degradation, plastic waste can be decomposed using physical processes, such as burial or burning, or chemical processes, such as photooxidative hydrolysis or decomposition with certain harsh chemicals. However, both physical and chemical methods have significant drawbacks (see Bano, K., et al., 2017. Current Pharmaceutical Biotechnology, 18(5), pp. 429-440).

[0004] Bio-based polyesters are an alternative to petroleum-based plastics. Polyhydroxyalkanoates (PHAs) are microbial polyesters with diverse structures synthesized by numerous prokaryotic microorganisms. PHAs are biocompatible, bioresorbable, and biodegradable, reducing their environmental impact. If left in the environment, PHA-based products degrade into CO2, H2O, and CH4, which promote natural cycles of recycling and regeneration. However, bacterial synthesis of PHAs is currently not cost-effective compared to petroleum-based plastics. Current technology uses a two-step microbial process: converting organic waste, including PHA waste, into volatile fatty acids (VFAs) under anaerobic conditions, followed by a second aerobic fermentation step to convert VFAs to PHAs (see Riaz, S., et al., 2021, Polymers, 13(2), p. 253).

[0005] Therefore, there is a need for new methods for decomposing plastic waste and also for producing PHAs.

[0006] [Summary of the Invention] The present inventors have developed a direct and efficient method for producing polyhydroxyalkanoates (PHAs) from polyester waste.

[0007] The inventors have demonstrated that this method enables the utilization of a wide variety of polyester monomers, including those that are difficult to biologically metabolize, such as 1,4-butanediol. This method enables the utilization of mixed polyester waste, including biodegradable polyesters (e.g., PHA, PHB, PHBH) and even non-biodegradable polyesters (e.g., PET).

[0008] The inventors have shown that the method can proceed via a one-step process in which a single microorganism is used to carry out the culture, as opposed to the two-step process currently used. The inventors have also shown that the culture can be carried out under aerobic or anaerobic conditions. This flexibility can reduce the need for oxygen in the fermenter, which is usually a limitation in large-scale fermentation.

[0009] We also identified microorganisms with suitable pathways for utilizing mixed polyester waste by constructing novel genomic metabolic models, and identified the genes involved in these pathways.

[0010] In one aspect, the present invention provides a method for producing polyhydroxyalkanoates (PHAs) from polyester waste, the method comprising the steps of: (a) providing a culture broth containing the polyester waste; and (b) culturing a microorganism in the culture broth to produce PHAs.

[0011] The microorganisms may produce PHAs using one or more polyester monomers from polyester waste. The microorganisms may produce PHAs using multiple polyester monomers from polyester waste. Preferably, the microorganisms produce PHAs using at least three, at least four, at least five, at least six, at least seven, or at least eight polyester monomers from polyester waste. Preferably, the microorganisms produce PHAs using polyester monomers from multiple polyesters from polyester waste. Preferably, the microorganisms produce PHAs using polyester monomers from at least three, at least four, at least five, at least six, at least seven, or at least eight polyesters from polyester waste. In some embodiments, the microorganisms produce PHAs using 1,4-butanediol from polyester waste.

[0012] Any suitable microorganism may be used in the method of the present invention. Preferably, the microorganism is from the genus Paracoccus. Preferably, the microorganism is Paracoccus denitrificans. Preferably, the microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof. Preferably, the microorganism is Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, Paracoccus denitrificans NBRC 102528, Paracoccus denitrificans NCCB 22021, Paracoccus denitrificans NBRC 13301, Paracoccus denitrificans NCIMB 8944, Paracoccus denitrificans DSM 15418, Paracoccus denitrificans DSM 415, Paracoccus denitrificans NCIMB 11627, Paracoccus denitrificans NCIMB 9722, Paracoccus denitrificans IMET 10380, Paracoccus denitrificans VKM B-1324, or Paracoccus denitrificans ICPB 3979.

[0013] The microorganism may contain genes encoding two or more pathways, three or more pathways, four or more pathways, five or more pathways, six or more pathways, or seven or more pathways selected from the following: (i) a pathway for succinate utilization; (ii) a pathway for lactic acid utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization. The microorganism can contain multiple genes encoding each of the following pathways: (i) a pathway for succinate utilization; (ii) a pathway for lactate utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyrate utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0014] Any suitable polyester waste may be utilized. Preferably, the polyester waste comprises two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol. Preferably, the polyester waste comprises succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol. In some embodiments, the polyester waste comprises 1,4-butanediol. Preferably, the polyester waste comprises polyester monomers in the form of free monomers. Preferably, the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters selected from polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), poly(ethylene adipate) (PEA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The polyester waste may be pretreated. Preferably, the polyester waste is mechanically and / or chemically treated.

[0015] The method of the present invention may further comprise a step of pretreating the polyester waste. Any suitable pretreatment may be used. Preferably, the method further comprises a step of mechanically treating the polyester waste (e.g., the polyester waste may be shredded). Preferably, the method further comprises a step of chemically treating the polyester waste (e.g., the polyester waste may be subjected to alkaline treatment).

[0016] Any suitable culture conditions may be used. Preferably, the culture broth contains the polyester waste in an amount of about 1 g / L to about 100 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 20 g / L, about 2 g / L to about 10 g / L, or about 2 g / L to about 5 g / L. Preferably, the culture broth contains a mineral salts medium. Preferably, the microorganism is cultured under aerobic or anaerobic conditions. In some embodiments, the microorganism is cultured under anaerobic conditions. Preferably, the microorganism is cultured for about 1 day to about 7 days, about 2 days to about 6 days, or about 3 days to about 5 days. Preferably, a single microbial strain is cultured. Preferably, the method includes a single culture step.

[0017] In some embodiments, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 75% by weight, or at least about 80% by weight of the polyester waste is utilized during cultivation. In some embodiments, at least about 0.01 mg / mL, at least about 0.02 mg / mL, at least about 0.03 mg / mL, at least about 0.04 mg / mL, at least about 0.05 mg / mL, or at least about 0.1 mg / mL of PHA is produced. In some embodiments, at least about 10 μg PHA / mg dry cell weight (DCW), at least about 20 μg PHA / mg DCW, at least about 30 μg PHA / mg DCW, at least about 40 μg PHA / mg DCW, or at least about 50 μg PHA / mg DCW is produced. The PHA may comprise or consist of polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof. In some embodiments, the PHA comprises or consists of polyhydroxybutyrate (PHB) or a copolymer thereof, hi some embodiments, the PHA comprises or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0018] The method may further comprise any other suitable steps. Preferably, the method further comprises the step of recovering the PHA.

[0019] In another aspect, the present invention provides a culture broth comprising polyester waste and a microorganism, wherein the microorganism is capable of utilizing a plurality of polyester monomers from the polyester waste to produce a PHA.

[0020] In another aspect, the present invention provides a polyhydroxyalkanoate (PHA) produced by a method according to the present invention.

[0021] In another aspect, the present invention provides an article comprising or consisting of a PHA produced by a method according to the present invention. The article may be packaging material.

[0022] In another aspect, the present invention provides the use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism is capable of utilizing multiple polyester monomers from the polyester waste to produce PHA.

[0023] In another aspect, the present invention provides a microorganism for producing polyhydroxyalkanoates (PHAs) from polyester waste, wherein the microorganism is capable of utilizing multiple polyester monomers from the polyester waste to produce PHA. The microorganism may be genetically engineered to express at least a portion of one or more of the pathways.

[0024] In another aspect, the present invention provides a vector comprising a gene encoding an enzyme for producing polyhydroxyalkanoate (PHA) from polyester waste.

[0025] In another aspect, the present invention provides a cell comprising a vector according to the invention.

[0026] Additional features and advantages of the present invention are described in, or will be apparent from, the following description of the presently preferred embodiments, which proceeds with reference to the drawings. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 is a schematic diagram showing the genetic competence-based microbial recycling of polyester plastic waste and their related monomers into polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans. [Figure 2A] Figure 2 visualizes the metabolic transformation of polyester monomers by Paracoccus denitrificans based on the constructed genome-scale model (GSM). The metabolic pathways identified for the conversion of different polyester monomers to PHA (PHB) by Paracoccus denitrificans and its associated genes and enzymes are shown. Metabolic pathways for the utilization of monomers from polymer building blocks: A) PBS (succinic acid), PLA (lactic acid), PHV (3-hydroxyvaleric acid), PBAT (adipic acid), hydroxycaproic acid, PHB (3-hydroxybutyric acid), and PBS (1,4 butanediol) are related to the central carbon metabolism (TCA cycle). Arrows also indicate the pathway for the production of PHB from the central carbon metabolism (TCA cycle). B) Metabolic pathways for the assimilation of PET (ethylene glycol). [Figure 2B]Figure 2 visualizes the metabolic transformation of polyester monomers by Paracoccus denitrificans based on the constructed genome-scale model (GSM). The metabolic pathways identified for the conversion of different polyester monomers to PHA (PHB) by Paracoccus denitrificans and its associated genes and enzymes are shown. Metabolic pathways for the utilization of monomers from polymer building blocks: A) PBS (succinic acid), PLA (lactic acid), PHV (3-hydroxyvaleric acid), PBAT (adipic acid), hydroxycaproic acid, PHB (3-hydroxybutyric acid), and PBS (1,4 butanediol) are related to the central carbon metabolism (TCA cycle). Arrows also indicate the pathway for the production of PHB from the central carbon metabolism (TCA cycle). B) Metabolic pathways for the assimilation of PET (ethylene glycol). [Figure 3] Figure 3 shows the biomass formation (cell mass dry weight) of Paracoccus denitrificans when different monomers were supplied as the sole carbon source at 0.3% (weight / volume) of the medium. [Figure 4] Figure 4 shows the amount of poly(3-hydroxybutyrate-co-valerate) (PHBV) produced by P. denitrificans using different plastic monomers as the sole carbon source. [Figure 5] FIG. 5 shows A) P. denitrificans cell growth and B) plastic monomer consumption and PHB production under anaerobic conditions. [Figure 6] FIG. 6 shows A) P. denitrificans cell growth and B) PHB production from mechanically and chemically pretreated polymers.

[0028] [Mode for Carrying Out the Invention] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example, it being understood that those skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0029] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," or "contains" and are all-inclusive, i.e., open-ended, and do not exclude additional, unrecited components, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0030] Numerical ranges are inclusive of the numbers defining the range. As used herein, the term "about" means approximately, in the region of, roughly, or in the vicinity of. Unless otherwise indicated, all nucleic acid sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0031] All publications mentioned herein are incorporated herein by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any publications cited herein should not be construed as an admission that these publications constitute prior art to the claims appended hereto.

[0032] a. Method for producing PHA from polymer waste In one aspect, the present invention provides a method for producing polyhydroxyalkanoates (PHAs) from polyester waste, the method comprising the steps of: (a) providing a culture broth containing the polyester waste; and (b) culturing a microorganism in the culture broth to produce PHAs.

[0033] a. Microorganisms for producing PHA from polyester waste Any suitable microorganism described herein (e.g., in the section entitled "Microorganisms") may be used to produce PHA from polyester waste. A mixture of microorganisms or a single microorganism may be used. In some embodiments, a single microorganism (e.g., a single microbial strain) is used.

[0034] The microorganisms may produce PHAs using one or more polyester monomers from the polyester waste. Preferably, the microorganisms produce PHAs using two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers from the polyester waste. The polyester monomers may be in the form of free polyester monomers, oligoesters, or polyesters. Preferably, the polyester monomers are in the form of free polyester monomers or oligoesters. Preferably, the polyester monomers are in the form of free polyester monomers.

[0035] In some embodiments, the microorganisms produce PHAs using one or more, two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers from polyester waste selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol. In some embodiments, the microorganisms produce PHAs using each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol from the polyester waste.

[0036] The microorganisms may produce PHAs using polyester monomers from one or more polyesters from the polyester waste, and preferably the microorganisms may produce PHAs using polyester monomers from two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters from the polyester waste.

[0037] In some embodiments, the microorganism is selected from the group consisting of polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), poly(ethylene adipate) (PEA The PHA is produced using polyester monomers from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters from polyester waste selected from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0038] In some embodiments, the microorganism produces PHAs utilizing polyester monomers from one or more, two or more, three or more, four or more, five or more, six or more, or seven polyesters selected from polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH). In some embodiments, the microorganism produces PHAs utilizing polyester monomers from one or more, two or more, or three polyesters selected from polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0039] In some embodiments, the microorganisms utilize at least about 20% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 75% by weight, at least about 80% by weight, at least about 85% by weight, at least about 90% by weight, at least about 95% by weight of the polyester waste.

[0040] b. Polyester waste Any suitable polyester waste may be used. As described above, the inventors have shown that this method enables the utilization of a wide variety of polyester monomers, including polyester monomers that are difficult to biologically metabolize, such as 1,4-butanediol. This method enables the utilization of mixed polyester waste, including biodegradable polyesters (e.g., PHA, PHB, PHBH) and even non-biodegradable polyesters (e.g., PET). The polyester waste may be polyester plastic waste.

[0041] Polyesters are polymers that contain an ester functional group in each repeat unit of the main chain. Polyesters can include naturally occurring polymers as well as synthetic polymers. While natural polyesters and a few synthetic polyesters are biodegradable, most synthetic polyesters are not. Polyesters include polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoates (PHAs) such as polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), polybutylene adipate terephthalate (PBAT), polyethylene furanoate (PEF), polycaprolactone (PCL), poly(ethylene adipate) (PEA), polybutylene succinate terephthalate (PBST), polyethylene succinate (PES), and poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL).

[0042] Polyesters are synthesized from polyester monomers. For example, PET can be synthesized from ethylene glycol and terephthalic acid; PTT can be synthesized from 1,3-propanediol and terephthalic acid; PBT can be synthesized from 1,4-butanediol and terephthalic acid; PLA can be synthesized from lactic acid; PHB can be synthesized from 3-hydroxybutyric acid; PHBV can be synthesized from 3-hydroxybutyric acid and 3-hydroxyvaleric acid; PBS can be synthesized from succinic acid and 1,4-butanediol; PBSA can be synthesized from succinic acid, 1,4-butanediol, and adipic acid; PB AT can be synthesized from 1,4-butanediol and adipic acid; PEF can be synthesized from 2,5-furandicarboxylic acid and ethylene glycol; PCL can be synthesized from 6-hydroxycaproic acid; PEA can be synthesized from adipic acid and ethylene glycol; PBST can be synthesized from succinic acid, terephthalic acid, and 1,4-butanediol; PES can be synthesized from ethylene glycol and succinic acid; and PBSTIL can be synthesized from succinic acid, lactic acid, 1,4-butanediol, terephthalic acid, and isophthalic acid. Conversely, polyester polymers can be decomposed into their polyester monomers, for example, by hydrolytic cleavage of ester bonds. Hydrolytic cleavage can occur passively or can be catalyzed by chemical or enzymatic processes.

[0043] Preferably, the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers. In a preferred embodiment, the polyester waste comprises a plurality of polyester monomers. The polyester monomers may be in the form of free polyester monomers, oligoesters, or polyesters. Preferably, the polyester monomers are in the form of free polyester monomers or oligoesters. Preferably, the polyester monomers are in the form of free polyester monomers. The inventors have shown that the method of the present invention can utilize either free polyester monomers, polyester oligomers, or polyester polymers (e.g., after a pretreatment step).

[0044] In some embodiments, the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol. In preferred embodiments, the polyester waste comprises 1,4-butanediol. In some embodiments, the polyester waste comprises each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol (in the form of free polyester monomers or in the form of polyester polymers).

[0045] Suitably, the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters. In a preferred embodiment, the polyester waste comprises a plurality of polyesters.

[0046] In some embodiments, the polyester waste is polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), poly The polyester may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters selected from poly(ethylene adipate) (PEA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), or copolymers thereof.

[0047] In some embodiments, the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, or seven polyesters selected from polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH). In some embodiments, the polyester waste comprises one or more, two or more, or three polyesters selected from polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0048] c. Pretreatment The polyester waste may be pretreated, i.e., treated before being added to the culture broth. The pretreated polyester waste may contain polyester monomers and / or polyester oligomers, particularly polyester monomers. The inventors have shown that pretreatment can promote the decomposition of polyester waste into smaller particles, making the polyester monomers more accessible. Any suitable pretreatment may be used (see, for example, Ragaert, K., et al., 2017. Waste Management, 69, pp. 24-58 and WO 2002036675(A2)). Any combination of pretreatment steps may be used, and each step may be repeated one or more times.

[0049] In some embodiments, the methods of the present invention include the steps of (a) pretreating polyester waste; (b) providing a culture broth containing the pretreated polyester waste; and (c) culturing a microorganism in the culture broth to produce PHAs. In some embodiments, the polyester waste is mechanically and / or chemically treated (prior to adding it to the culture broth).

[0050] In some embodiments, the polyester waste is mechanically processed (prior to being added to the culture broth). For example, the polyester waste may be separated and / or sorted; bailed; washed; crushed, shredded, and / or chopped; and / or compounded and / or pelleted before being added to the culture broth.

[0051] In some embodiments, the polyester waste is separated and / or sorted (before being added to the culture broth). Separation and / or sorting can be based on shape, density, size, color, or chemical composition. The polyester waste may be sorted manually or automatically (e.g., by flotation). In some embodiments, the polyester waste is bailed (before being added to the culture broth). If plastic processing is not performed at the location where the plastics are sorted, the plastics are often baled for transportation purposes between locations. In some embodiments, the polyester waste is washed (before being added to the culture broth). Washing can be used to remove contaminants, such as organic contaminants.

[0052] In a preferred embodiment, the polyester waste is crushed, shredded, and / or chopped (before being added to the culture broth). This can reduce the size of the polyester waste, for example, to produce flakes. In some embodiments, the polyester waste is shredded (before being added to the culture broth). Preferably, the polyester waste has a particle size of about 0.1 mm to about 20 mm, about 0.2 mm to about 10 mm, about 0.3 mm to about 5 mm, about 0.4 mm to about 2 mm, or about 0.5 mm to about 1 mm. Preferably, the polyester waste has a particle size of about 100 μm to about 5000 μm, about 200 μm to about 4000 μm, about 300 μm to about 3000 μm, about 400 μm to about 2000 μm, or about 500 μm to about 1000 μm.

[0053] In some embodiments, the polyester waste is chemically treated (before being added to the culture broth). Chemical treatment can be used to partially or completely depolymerize the polyester into its polyester monomers. Depending on the chemical agent and the polyester, different depolymerization pathways may be used, such as methanolysis, glycolysis, hydrolysis, ammonolysis, aminolysis, and / or hydrogenation. In some embodiments, the polyester waste is hydrolyzed (before being added to the culture broth). During hydrolysis, the polyester may react with water under neutral or acidic conditions to cleave the polyester chains. High temperature and / or high pressure can be used to promote hydrolysis. In some embodiments, the polyester waste is subjected to an alkaline treatment (before being added to the culture broth). Preferably, the polyester waste can be incubated in an alkaline solution (e.g., about 0.5 M to about 2 M NaOH or about 1 M to about 2 M NaOH) at about 30°C to about 40°C (e.g., about 37°C) for about 5 days to about 20 days (e.g., about 7 days to about 15 days), optionally with shaking or stirring at about 200 rpm to about 500 rpm (e.g., about 300 rpm to about 400 rpm). Preferably, the polyester waste is neutralized after the alkaline treatment. Preferably, the polyester waste is neutralized with an acidic solution (e.g., containing hydrochloric acid). Preferably, the polyester waste is neutralized to about pH 7.

[0054] In some embodiments, the polyester waste is mechanically and chemically treated (prior to being added to the culture broth), hi some embodiments, the polyester waste is crushed, shredded, and / or chopped and subjected to alkaline treatment (prior to being added to the culture broth).

[0055] d. Culture broth In one aspect, the present invention provides a culture broth comprising polyester waste and a microorganism. The microorganism may be any microorganism described herein (e.g., in the section entitled "Microorganisms") that is suitable for producing PHA from polyester waste. The polyester waste may be any polyester waste described herein (e.g., in the subsection entitled "Polyester Waste"). Preferably, the culture broth may further comprise a PHA, preferably any PHA described herein (e.g., in the subsection entitled "Production of PHA").

[0056] The polyester waste may be added to the culture broth in any suitable amount. Preferably, the culture broth contains the polyester waste in an amount of at least about 1 g / L, at least about 2 g / L, at least about 3 g / L, at least about 4 g / L, or at least about 5 g / L. Preferably, the culture broth contains the polyester waste in an amount of about 100 g / L or less, about 90 g / L or less, about 80 g / L or less, about 70 g / L or less, about 60 g / L or less, about 50 g / L or less, about 40 g / L or less, about 30 g / L or less, about 25 g / L or less, about 20 g / L or less, about 15 g / L or less, about 10 g / L or less, about 9 g / L or less, about 8 g / L or less, about 7 g / L or less, about 6 g / L or less, or about 5 g / L or less. Preferably, the culture broth contains the polyester waste in an amount of about 1 g / L to about 100 g / L, about 1 g / L to about 90 g / L, about 1 g / L to about 80 g / L, about 1 g / L to about 70 g / L, about 1 g / L to about 60 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 25 g / L, about 1 g / L to about 20 g / L, about 2 g / L to about 10 g / L, or about 2 g / L to about 5 g / L.

[0057] Any suitable culture broth may be used to culture the microorganism. Preferably, the culture broth has a pH of about 7 and contains all nutrients and trace elements necessary for culturing the microorganism. The culture broth may vary depending on the microorganism and / or culture conditions used. For example, the optimal culture broth for anaerobic growth of Paracoccus denitrificans is described in Hahnke, SM, et al., 2014. Frontiers in microbiology, 5, p. 18.

[0058] In some embodiments, the culture broth comprises a mineral salts medium. Preferably, the culture broth comprises the mineral salts medium in an amount of at least about 80% (volume / volume), at least about 85% (volume / volume), at least about 90% (volume / volume), or at least about 95% (volume / volume). Preferably, the mineral salts medium may comprise about 22.7 g / L dipotassium hydrogen phosphate, about 0.95 g / L potassium dihydrogen phosphate, about 0.67 g / L ammonium sulfate, and about 2 mL / L of a trace metals solution. Preferably, the trace metals solution may comprise sodium, zinc, calcium, iron, molybdenum, copper, cobalt, manganese, and magnesium.

[0059] e.Culture conditions Any suitable culture conditions may be used. As mentioned above, the present inventors have shown that the present method can proceed through a one-step process in which a single microorganism is used to carry out the culture. The present inventors have also shown that the culture can be carried out under aerobic or anaerobic conditions. The culture conditions may vary depending on the microorganism used.

[0060] Preferably, the microorganisms are cultured under aerobic or anaerobic conditions. As used herein, "aerobic" conditions are rich in free oxygen (O). As used herein, "anaerobic" conditions are characterized by limited or no free oxygen and the inclusion of other electron acceptors (e.g., ammonium sulfate, potassium nitrate, and / or sodium sulfate). Preferably, anaerobic conditions can be created by replacing oxygen with another gas, such as an inert gas (e.g., helium). Preferably, anaerobic conditions can be created by replacing with helium (helium washing). In some embodiments, the microorganisms are cultured under aerobic conditions. In some embodiments, the microorganisms are cultured under anaerobic conditions. The microorganisms can be cultured partially under aerobic conditions and partially under anaerobic conditions.

[0061] The microorganisms may be cultured for any suitable period of time. Preferably, the microorganisms are cultured for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, or at least about 5 days. Preferably, the microorganisms are cultured for about 10 days or less, about 9 days or less, about 8 days or less, about 7 days or less, about 6 days or less, or about 5 days or less. Preferably, the microorganisms are cultured for about 1 to about 7 days, about 2 to about 6 days, or about 3 to about 5 days. Preferably, the microorganisms are cultured until a cell biomass of at least about 0.1 mg / mL, at least about 0.2 mg / mL, at least about 0.3 mg / mL, at least about 0.4 mg / mL, or at least about 0.5 mg / mL is reached.

[0062] Microorganisms may be cultured at any suitable temperature. For example, Paracoccus denitrificans can be cultured at temperatures between 11°C and 45°C (Hahnke, SM, et al., 2014. Frontiers in Microbiology, 5, p. 18). Preferably, microorganisms are cultured at about 30°C to about 40°C. Preferably, microorganisms are cultured at about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, or about 37°C. Preferably, microorganisms are cultured at about 30°C, about 34°C, or about 37°C.

[0063] Microorganisms can be cultured under static conditions, shaking conditions, or stirring conditions. In some embodiments, microorganisms are cultured under shaking or stirring conditions. In some embodiments, microorganisms are cultured under shaking conditions. Any suitable shaking conditions may be used (see, for example, Kloeckner, W. and Buchs, J., 2012. Trends in biotechnology, 30(6), pp. 307-314). Preferably, the shaking conditions may be about 100 rpm to about 400 rpm, or about 200 rpm to about 300 rpm.

[0064] The culture broth may be inoculated with a microorganism. Any suitable inoculum may be used. Preferably, the inoculum is added in an amount of at least about 1% (volume / volume) or at least about 2% (volume / volume). Preferably, the inoculum is added in an amount of about 1% (volume / volume) or about 2% (volume / volume). Preferably, the inoculum may be derived from culturing the microorganism in a rich medium (e.g., LB medium).

[0065] The method may include one or more culturing steps. In some embodiments, the method includes a single culturing step. In some embodiments, the method does not include a second culturing step with a second microorganism (or a second mixture of microorganisms). During a single culturing step, the culture conditions may be adjusted, for example, to lower or raise the temperature, and / or the culture conditions may be monitored and maintained, for example, to maintain minimum pH and / or nutrient levels. A single culturing step may consist of culturing a single microorganism (or a single mixture of microorganisms) in the culture broth. A further culturing step may consist of culturing another microorganism (or another mixture of microorganisms) in the culture broth.

[0066] f. PHA production The type and / or amount of PHA produced may vary depending, for example, on the polyester waste material, the microorganism, and / or the culture conditions utilized, and one skilled in the art would be able to optimize the method to achieve the desired type and / or amount of PHA. Polyhydroxyalkanoates (PHAs) comprise a group of natural biodegradable polyesters synthesized by microorganisms and may have the following general molecular formula, where x=1-8 and n=100-1000s (see Li, Z., et al., 2016. NPG Asia Materials, 8(4), pp. e265-e265):

[0067] [ka]

[0068] PHAs are the largest group of natural polyesters, with over 150 different PHA monomers identified. Commonly synthesized PHA monomers include 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. Exemplary PHAs include poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), and poly(3-hydroxyoctanoate) (PHO), poly(3-hydroxynonanoate) (PHN), and copolymers thereof with 3-hydroxyhexanoate (HHx), 3-hydroxyheptanoate (HH), and / or 3-hydroxydecanoate (HD).

[0069] The PHA produced by the present invention will vary depending on the polyester waste. For example, if the polyester waste is rich in polyester monomers such as lactic acid, succinic acid, ethylene glycol, adipic acid, 3-hydroxybutyric acid, 6-hydroxycaproic acid, and / or 1,4-butanediol, the PHA produced may be rich in 3-hydroxybutyric acid monomers. For example, if the polyester waste is rich in 3-hydroxyvaleric acid, the PHA produced may be rich in 3-hydroxyvalerate monomers.

[0070] The PHA produced by the present invention may comprise or consist of a PHA comprising 3-hydroxybutyrate monomers, 3-hydroxyvalerate monomers, and / or 3-hydroxyhexanoate monomers. The PHA produced by the present invention may comprise or consist of a PHA comprising 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers. The PHA produced by the present invention may comprise or consist of a PHA comprising 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers. The PHA produced by the present invention may comprise 3-hydroxybutyrate monomers. In some embodiments, the PHA produced by the present invention comprises or consists of about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxybutyrate monomers and about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxyvalerate monomers. In other embodiments, for example, when polyester waste is rich in 3-hydroxyvaleric acid, the PHA produced by the present invention comprises or consists of about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxybutyrate monomers and about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxyvalerate monomers.

[0071] The PHA produced by the present invention may comprise or consist of poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The PHA produced by the present invention may comprise or consist of poly(3-hydroxybutyrate) (PHB) or a copolymer thereof and / or poly(3-hydroxyvalerate) (PHV) or a copolymer thereof. The PHA produced by the present invention may comprise or consist of poly(3-hydroxybutyrate) (PHB) or a copolymer thereof. The PHB copolymer may comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and / or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0072] The PHA produced by the present invention can comprise or consist of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In some embodiments, the PHBV comprises or consists of about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxybutyrate and about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxyvalerate. In other embodiments, the PHBV comprises or consists of about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxybutyrate monomers and about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxyvalerate monomers.

[0073] The methods of the invention can be used to produce PHA in amounts of at least about 0.01 mg / mL, at least about 0.02 mg / mL, at least about 0.03 mg / mL, at least about 0.04 mg / mL, at least about 0.05 mg / mL, or at least about 0.1 mg / mL. The methods of the invention can be used to produce PHA in amounts of at least about 10 μg PHA / mg dry cell weight (DCW), at least about 20 μg PHA / mg DCW, at least about 30 μg PHA / mg DCW, at least about 40 μg PHA / mg DCW, or at least about 50 μg PHA / mg DCW.

[0074] g. PHA recovery, separation, and / or purification After the cultivation step is completed, the PHAs may be recovered, separated, and / or purified from the resulting microbial slurry using any suitable method known in the art (see, e.g., Pagliano, G., et al., 2021. Frontiers in Bioengineering and Biotechnology, 9, p. 54; Perez-Rivero, C., et al., 2019. Biochemical Engineering Journal, 150, p. 107283; and Lopez-Abelairas, M., et al., 2015. Biochemical Engineering Journal, 93, pp. 250-259).

[0075] In some embodiments, the method of the present invention comprises the steps of (a) providing a culture broth containing polyester waste; (b) culturing microorganisms in the culture broth to produce a microbial slurry containing PHAs; and (c) recovering the PHAs from the microbial slurry. In some embodiments, the method of the present invention comprises the steps of (a) pretreating polyester waste; (b) providing a culture broth containing the pretreated polyester waste; (c) culturing microorganisms in the culture broth to produce a microbial slurry containing PHAs; and (d) recovering the PHAs from the microbial slurry. The PHAs may be recovered by solvent and / or cell lysis.

[0076] Preferably, PHA can be recovered from the microbial slurry by solvent extraction (see, for example, Pagliano, G., et al., 2021. Frontiers in Bioengineering and Biotechnology, 9, p. 54). Solvent extraction of PHA from the microbial slurry can include the following steps: (i) contacting and mixing the microbial slurry with a solvent; (ii) heating the mixture; (iii) separating the extraction residue (non-PHA biomass and water) from the PHA-enriched phase (PHA dissolved in the solvent); and (iv) separating the PHA from the solvent by evaporation of the PHA precipitate.

[0077] Preferably, the PHA can be recovered from the microbial slurry by cell lysis (see, for example, Pagliano, G., et al., 2021. Frontiers in Bioengineering and Biotechnology, 9, p. 54). Cell lysis can include the following steps: (i) mixing the microbial slurry with additives (e.g., alkali, surfactant, oxidizing agent) to solubilize non-PHA components; (ii) separating the solid PHA from the liquid (containing the non-PHA components); and (iii) drying and purifying the PHA.

[0078] After recovery, the PHA may be subjected to one or more further downstream processing steps, such as separation and / or purification (see, for example, Perez-Rivero, C., et al., 2019. Biochemical Engineering Journal, 150, p. 107283). In some embodiments, the PHA is separated. Conventional separation methods include sedimentation, crystallization, centrifugation, decantation, filtration, or a combination thereof. In some embodiments, the PHA is purified. Suitably, the PHA may be purified by washing the crude PHA with a solvent, such as ethanol, acetone, diethyl ether, or any combination thereof.

[0079] In some embodiments, the method of the present invention comprises the steps of: (a) providing a culture broth containing polyester waste; (b) culturing microorganisms in the culture broth to produce a microbial slurry containing PHA; (c) recovering the PHA from the microbial slurry to obtain crude PHA; and (d) separating and / or purifying the crude PHA to obtain purified PHA. In some embodiments, the method of the present invention comprises the steps of: (a) pretreating polyester waste; (b) providing a culture broth containing the pretreated polyester waste; (c) culturing microorganisms in the culture broth to produce a microbial slurry containing PHA; (d) recovering the PHA from the microbial slurry to obtain crude PHA; and (e) separating and / or purifying the crude PHA to obtain purified PHA.

[0080] b. PHA and goods In one aspect, the present invention provides a polyhydroxyalkanoate (PHA) obtained or obtainable by a method according to the present invention.

[0081] The PHA can comprise or consist of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). PHBV is one of the most promising biopolymers for replacing petroleum-based plastics, but its low processing productivity and high selling price are major barriers to its widespread use. In some embodiments, the PHBV comprises or consists of about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxybutyrate and about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxyvalerate. In other embodiments, the PHBV comprises or consists of about 5% to about 10% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxybutyrate monomers and about 90% to about 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxyvalerate monomers.

[0082] The PHA may be provided in any suitable form, for example, the PHA may be provided in the form of a resin, sealant, adhesive, granules, powder, microbeads, spheres, sheets, films, pellets, and the like.

[0083] In one aspect, the present invention provides an article comprising or consisting of a polyhydroxyalkanoate (PHA) produced by a method according to the present invention.

[0084] PHAs can have a wide range of applications, such as packaging materials for food and other perishable goods (see, for example, Bugnicourt, E., et al., 2014, eXPRESS Polymer Letters Vol. 8, No. 11 (2014) 791-808); medical and pharmaceutical fields (see, for example, Valappil, SP, et al., 2006, Expert Review of Medical Devices, 3 (6), pp. 853-868); and the transportation sector, such as automobiles or aircraft. Applications include packaging materials, molded articles, paper coatings, nonwoven fabrics, adhesives, films, and functional additives.

[0085] Preferably, the PHA produced by the method according to the present invention can be used as packaging materials, plastic bags, cutlery, and food containers. In some embodiments, the article of the present invention is a packaging material, film, and / or bag. For example, the article of the present invention can be a food packaging material, fresh film, mulch film, laminating film, wrapping film, heat-shrinkable film, shopping bag, garbage bag, gift bag, and produce bag. In some embodiments, the article of the present invention is a vial, bottle, or container.

[0086] c. Microorganisms In one aspect, the present invention provides a microorganism for producing polyhydroxyalkanoates (PHAs) from polyester waste. The microorganism may be isolated from the microorganism's natural environment or may be produced by technological processes (e.g., genetic engineering).

[0087] As used herein, "microbe" or "microorganism" may refer to a microscopic-sized organism that may exist in its single-cell form or as a colony of cells. Exemplary microorganisms include bacteria, archaea, fungi, and protists.

[0088] Preferably, the microorganism of the present invention may be a bacterium. Preferably, the microorganism of the present invention is from the family Rhodobacteraceae. Preferably, the microorganism of the present invention is from the genus Paracoccus. Species of the genus Paracoccus include P. acridae, P. aeridis, P. aerius, P. aestuarii, P. aestuariivivens, P. alcaliphilus, P. alimentarius, P. alkanivorans, and P. alkeni. fer), P. aminophilus, P. aminovorans, P. amoyensis, P. angustae, P. aquimaris, P. aurantiacus, P. baruchii, P. beibuensis, P. binzhouensis, P. bogoriensi P. bogoriensis, P. caeni, P. carotinifaciens, P. cavernae, P. chinensis, P. communis, P. contaminans, P. denitrificans, P. endophyticus, P. ferrooxidans xidans, P. fistulariae, P. fontiphilus, P. gahaiensis, P. haematequi, P. haeundaensis, P. halophilus, P. halotolerans, P. hibisci, P. hibiscisoli, P.P.homiensis, P.huijuniae, P.indicus, P.isoporae, P.jeotgali, P.kamogawaensis, P.kawasakiensis, P.kocurii, P.kondratievae, P.koreensis, P.laevglucosivorans iglucosivorans, P. liaowanqingii, P. lichenicola, P. limosus, P. litorisediminis, P. luteus, P. lutimaris, P. mangrovi, P. marcusii, P. marinus, P. methylutens, P. mutanolyticus mutanolyticus, P. niistensis, P. nototheniae, P. oceanense, P. onubensis, P. pacificus, P. panacisoli, P. pantotrophus, P. pueri, P. ravus, P. rhizosphaerae, P. salipardis salipaludis, P. saliphilus, P. sanguinis, P. sediminilitris, P. sediminis, P. seriniphilus, P. shandongensis, P. siganidrum, P. simplex, P. solventivorans, P. sorzidisorisordidisoli, P. speluncae, P. sphaerophysae, P. stylophorae, P. subflavus, P. sulfuroxidans, P. suum, P. tegillarcae, P. thiocyanatus Mention may be made of P. iocyanatus, P. thiophilus, P. tibetensis, P. versutus, P. xiamenensis, P. yeei, P. zeaxanthinifaciens, and P. zhejiangensis.

[0089] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans, Paracoccus pantotrophus, or Paracoccus belustus. In some embodiments, the microorganism of the present invention is Paracoccus denitrificans or Paracoccus pantotrophus.

[0090] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans, a Gram-negative, non-motile, denitrifying (nitrate-reducing) cocci (Kelly, DP et al., 2006. International Journal of Systematic and Evolutionary Microbiology, 56(10), pp. 2495-2500).

[0091] In some embodiments, the microorganism of the present invention is selected from the group consisting of Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, Paracoccus denitrificans NBRC 102528, Paracoccus denitrificans NCCB 22021, Paracoccus denitrificans NBRC 13301, Paracoccus denitrificans NCIMB 8944, Paracoccus denitrificans DSM 15418, Paracoccus denitrificans DSM 415, Paracoccus denitrificans NCIMB 11627, Paracoccus denitrificans NCIMB 9722, Paracoccus denitrificans IMET 10380, Paracoccus denitrificans VKM B-1324, Paracoccus denitrificans ICPB 3979, or a derivative thereof.

[0092] In some embodiments, the microorganism of the present invention is selected from the group consisting of Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, Paracoccus denitrificans NBRC 102528, Paracoccus denitrificans NCCB 22021, or a derivative thereof.

[0093] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, or a derivative thereof.

[0094] In a preferred embodiment, the microorganism of the present invention is Paracoccus denitrificans DSM 413 or a derivative thereof. Paracoccus denitrificans DSM 413 was deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) before August 22, 1990, and is the type strain. Derivatives of Paracoccus denitrificans DSM 413 include Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, and Paracoccus denitrificans NBRC. 102528, and Paracoccus denitrificans NCCB 22021.

[0095] Comparison of 16S rRNA gene sequences is routinely used to confirm the precise location of Paracoccus strains within the alpha-3 subgroup of the Alphaproteobacteria class (Kelly, DP et al., 2006. International Journal of systematic and evolutionary microbiology, 56(10), pp. 2495-2500). Preferably, the 16S rRNA gene sequence of the microorganism has at least 95%, at least 96%, at least 97%, at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100.0% identity to the 16S rRNA gene sequence of Paracoccus denitrificans DSM 413 (see, e.g., GenBank Accession No. Y16929.1).

[0096] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans PD1222 or a derivative thereof. Paracoccus denitrificans PD1222 (NCBI: txid318586) is a derivative of DSM 413 and is a model soil microorganism capable of carrying out the complete denitrification pathway (Baker, SC, et al., 1998. Microbiology and Molecular Biology Reviews, 62(4), pp. 1046-1078). Paracoccus denitrificans PD1222 may also be known as Paracoccus denitrificans NCCB 97099 (Kelly, DP et al., 2006. International Journal of Systematic and Evolutionary Microbiology, 56(10), pp. 2495-2500).

[0097] In some embodiments, the microorganism of the invention is Paracoccus denitrificans deposited by SOCIETEDES PRODUITS NESTLE at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15) under the terms of the Budapest Treaty on September 12, 2022, under the number CNCM I-5881. The deposited strain may be referred to herein as Paracoccus denitrificans CNCM I-5881.

[0098] In some embodiments, the microorganism of the invention has at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100.0% sequence identity to Paracoccus denitrificans having GenBank assembly accession number GCA_000203895.1.

[0099] As used herein, a "derivative" of an existing strain can refer to a genetically engineered variant (e.g., a variant in which one or more genes have been knocked out and / or inserted) or a naturally occurring variant (e.g., a variant that has undergone genetic drift). Typically, a derivative strain can have at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100.0% sequence identity to the original strain.

[0100] a. Utilization of polyester waste The microorganisms of the present invention may be capable of producing PHAs using one or more polyester monomers, preferably two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers.

[0101] In some embodiments, the microorganism is capable of producing PHAs utilizing one or more, two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol. In some embodiments, the microorganism is capable of producing PHAs utilizing each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

[0102] The microorganism may comprise genes encoding one or more pathways for polyester monomer utilization. Suitably, the microorganism comprises genes encoding two or more pathways, three or more pathways, four or more pathways, five or more pathways, six or more pathways, or seven or more pathways for polyester monomer utilization.

[0103] In some embodiments, the microorganism comprises genes encoding two or more pathways, three or more pathways, four or more pathways, five or more pathways, six or more pathways, or seven or more pathways selected from: (i) a pathway for succinate utilization; (ii) a pathway for lactate utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0104] In some embodiments, the microorganism comprises multiple genes encoding each of the following pathways: (i) a pathway for succinate utilization; (ii) a pathway for lactate utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyrate utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0105] In a preferred embodiment, the microorganism comprises genes encoding a pathway for 1,4-butanediol utilization. Suitably, the microorganism comprises one or more genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase. In some embodiments, the microorganism contains (i) a gene encoding a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31; (ii) a gene encoding a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33; or (iii) a gene encoding a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% to the amino acid sequence of SEQ ID NO: 23. , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35; (iv) a gene encoding an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35; (v) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37;(vi) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5; (vii) a gene encoding a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39; (viii) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4 and (ix) a gene encoding a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43. In some embodiments, the microorganism comprises (i) a gene encoding a methanol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 32; (ii) a gene encoding a methanol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 34;(iii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 24; (iv) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, or at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 36. (v) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38; (vi) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38; (vii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:6; (vii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:40; (viii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:42;and (ix) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:44;

[0106] In some embodiments, the microorganism comprises a gene encoding a pathway for succinate utilization. Preferably, the microorganism comprises one or more genes encoding a succinate dehydrogenase. In some embodiments, the microorganism comprises (i) a gene encoding a succinate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the microorganism comprises (i) a gene encoding a succinate dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 2.

[0107] In some embodiments, the microorganism contains a gene encoding a pathway for lactic acid utilization. Preferably, the microorganism contains one or more genes encoding D-lactate dehydrogenase. In some embodiments, the microorganism contains (i) a gene encoding a D-lactate dehydrogenase that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the microorganism contains (i) a gene encoding a D-lactate dehydrogenase that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:4.

[0108] In some embodiments, the microorganism comprises genes encoding a pathway for ethylene glycol utilization. Preferably, the microorganism comprises one or more genes encoding an alcohol dehydrogenase, an aldehyde dehydrogenase, and a glyoxylate reductase. In some embodiments, the microorganism comprises (i) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5; (ii) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7; , a gene encoding an aldehyde dehydrogenase having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9; and (iii) a gene encoding a glyoxylate reductase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.In some embodiments, the microorganism contains (i) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:6; (ii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:8. and (iii) a gene encoding a glyoxylate reductase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 10.

[0109] In some embodiments, the microorganism comprises genes encoding a pathway for adipic acid utilization. Preferably, the microorganism comprises one or more genes encoding a long-chain fatty acid CoA ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxybutyryl-CoA dehydrogenase, and a 3-oxoadipyl-CoA thiolase. In some embodiments, the microorganism contains (i) a gene encoding a long-chain fatty acid CoA ligase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:11; (ii) a gene encoding an acyl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:13; or (iii) a gene encoding an acyl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least (iv) a gene encoding an enoyl-CoA hydratase having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17; (iv) a gene encoding a 3-hydroxybutyryl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17; and (v) a gene encoding a 3-oxoadipyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19.In some embodiments, the microorganism contains (i) a gene encoding a long-chain fatty acid CoA ligase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:12; (ii) a gene encoding an acyl-CoA dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:14; or (iii) a gene encoding an enoyl-CoA hydratase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:16. (iv) a gene encoding 3-hydroxybutyryl-CoA dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 18; and (v) a gene encoding 3-oxoadipyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0110] In some embodiments, the microorganism comprises genes encoding a pathway for 6-hydroxycaproic acid utilization. Preferably, the microorganism comprises one or more genes encoding an alcohol dehydrogenase and an aldehyde dehydrogenase. In some embodiments, the microorganism comprises (i) a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21; and / or (ii) a gene encoding an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, the microorganism comprises (i) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:22; and / or (ii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:24.

[0111] In some embodiments, the microorganism comprises genes encoding a pathway for 3-hydroxybutyrate utilization. Preferably, the microorganism comprises one or more genes encoding an acyl-CoA synthetase and a 3-hydroxybutyrate dehydrogenase. In some embodiments, the microorganism comprises (i) a gene encoding an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25; and / or (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, the microorganism comprises (i) a gene encoding an acyl-CoA synthetase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:26; and / or (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:28.

[0112] In some embodiments, the microorganism comprises genes encoding a pathway for 3-hydroxyvalerate utilization. Preferably, the microorganism comprises one or more genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and 3-ketoacyl-CoA thiolase. In some embodiments, the microorganism comprises (i) a gene encoding an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25; (ii) a gene encoding an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% sequence identity to the amino acid sequence of SEQ ID NO:27; and (iii) a gene encoding a 3-hydroxybutyrate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:29.In some embodiments, the microorganism contains (i) a gene encoding an acyl-CoA synthetase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:26; (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:28. and (iii) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:30.

[0113] A microorganism may contain genes encoding one or more pathways for PHA synthesis. Currently, a total of at least 14 pathways leading to PHA synthesis have been reported. Preferably, the microorganism contains one or more genes encoding a PHA synthase. PHA synthase is a key enzyme involved in PHA biosynthesis and functions by polymerizing monomeric hydroxyalkanoate substrates. PHA synthases have been classified into four major classes based on their primary sequence, substrate specificity, and subunit composition. Any suitable PHA synthase, such as a natural PHA synthase or a genetically engineered PHA synthase, may be used (Chek, MF, et al., 2017, Scientific Reports, 7(1), pp. 1-15).

[0114] Preferably, the microorganism comprises one or more genes encoding 3-ketoacyl-CoA thiolase, acetoacetyl-CoA reductase, and PHA synthase. Preferably, the microorganism comprises one or more genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and PHA synthase.

[0115] In some embodiments, the microorganism has (i) a gene encoding a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:45; (ii) a gene encoding a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100% sequence identity to the amino acid sequence of SEQ ID NO:47; and (iii) a gene encoding an enoyl-CoA hydratase having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:49; and (iv) a gene encoding a PHA synthase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, the microorganism contains (i) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:46; (ii) a gene encoding an enoyl-CoA hydratase and having at least 70%, at least 75%, at least 80%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:48. and (iii) a gene encoding a PHA synthase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:50.

[0116] The microorganisms of the invention may be capable of producing PHAs using polyester monomers from one or more polyesters, preferably two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters.

[0117] In some embodiments, the microorganism is polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), poly(ethylene adipate) (PEA), polylactic acid ( PHAs can be produced using polyester monomers from one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more polyesters selected from PBS, PBSA, PBST, PBSTIL, PBT, PBAT, PET, PEA, PLA, PCL, PHB, PHBV, and PHBH. In some embodiments, the microorganism can produce PHAs using polyester monomers from each of PBS, PBSA, PBST, PBSTIL, PBT, PBAT, PET, PEA, PLA, PCL, PHB, PHBV, and PHBH.

[0118] B genetically engineered microorganisms In some embodiments, the microorganism is genetically engineered. The microorganism can be genetically engineered to improve utilization of one or more polyester monomers and / or to improve synthesis of PHA. This can be achieved, for example, by introducing genes encoding part or all of the pathway, or by optimizing the promoter and / or RBS to increase expression of part or all of the pathway (see, for example, Zhang, X., et al., 2020. Trends in biotechnology, 38(7), pp. 689-700). In some embodiments, the microorganism is genetically engineered to overexpress all or part of the pathway.

[0119] The microorganism may be genetically engineered by any suitable method (see, for example, Keasling, JD, 1999. Trends in biotechnology, 17(11), pp. 452-460 and Yan, Q. and Fong, SS, 2017. Frontiers in microbiology, 8, p. 2060). Preferably, the microorganism is genetically engineered by transfection, transduction, or gene editing.

[0120] In some embodiments, the microorganism is genetically engineered by transfection. The terms "transfection" or "transformation" can refer to a type of genetic engineering in which a non-viral vector is used to deliver a gene to a target cell. A typical transformation method for bacteria can use plasmid DNA.

[0121] In some embodiments, microorganisms are genetically engineered by transduction. The term "transduction" can refer to a type of genetic engineering in which a viral vector is used to deliver a gene to a target cell. A typical transduction method for bacteria can use a bacteriophage.

[0122] In some embodiments, microorganisms are genetically engineered by gene editing. The term "gene editing" can refer to a type of genetic manipulation in which nucleic acids are inserted, deleted, or replaced within a cell. Gene editing can be achieved using engineered nucleases that can be targeted to desired sites in a polynucleotide (e.g., a genome). Such nucleases can create site-specific double-strand breaks at the desired locations, which can then be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations. Such nucleases can be delivered to target cells using vectors. Examples of suitable nucleases known in the art include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas systems (see, e.g., Gaj, T. et al. (2013) Trends Biotechnol. 31:397-405).

[0123] In some embodiments, the microorganism has been genetically engineered to express, enhance expression, and / or overexpress at least a portion of one or more of the pathways described. In some embodiments, the microorganism has been genetically engineered to express, enhance expression, and / or overexpress at least a portion of one or more pathways for polyester monomer utilization and / or the microorganism has been genetically engineered to express, enhance expression, and / or overexpress at least a portion of one or more pathways for PHA synthesis.

[0124] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for succinate utilization. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding succinate dehydrogenase. In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress a succinate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the microorganism is genetically engineered to introduce a gene encoding a succinate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:2.

[0125] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for lactate utilization. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding D-lactate dehydrogenase. In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress a D-lactate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the microorganism has been genetically engineered to introduce a gene encoding D-lactate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:4.

[0126] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for ethylene glycol utilization. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding an alcohol dehydrogenase, an aldehyde dehydrogenase, and / or a glyoxylate reductase. In some embodiments, the microorganism has (i) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5; (ii) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7; and (iii) a glyoxylate reductase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.In some embodiments, the microorganism contains (i) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:6; (ii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:8; and (iii) a gene encoding a glyoxylate reductase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:10.

[0127] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for adipic acid utilization. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding long-chain fatty acid CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxybutyryl-CoA dehydrogenase, and / or 3-oxoadipyl-CoA thiolase.In some embodiments, the microorganism has (i) a long-chain fatty acid CoA ligase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:11; (ii) an acyl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:13; or (iii) an acyl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:15. (iv) an enoyl-CoA hydratase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:17; and (v) a 3-oxoadipyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:19.In some embodiments, the microorganism contains (i) a gene encoding a long-chain fatty acid CoA ligase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 12; (ii) a gene encoding an acyl-CoA dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 14; or (iii) a gene encoding an enoyl-CoA hydratase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% to the nucleotide sequence of SEQ ID NO: 16. , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:18; (iv) a gene encoding 3-hydroxybutyryl-CoA dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:18; and (v) a gene encoding 3-oxoadipyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:20.

[0128] In some embodiments, the microorganism is genetically engineered to express, enhance expression, and / or overexpress at least a portion of a pathway for 6-hydroxycaproic acid utilization. Preferably, the microorganism is genetically engineered to express, enhance expression, and / or overexpress one or more genes encoding alcohol dehydrogenase and / or aldehyde dehydrogenase. In some embodiments, the microorganism is genetically engineered to express, enhance expression of, and / or overexpress (i) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:21; and / or (ii) an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:23. In some embodiments, the microorganism has been genetically engineered to introduce (i) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:22; and / or (ii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:24.

[0129] In some embodiments, the microorganism is genetically engineered to express, enhance expression, and / or overexpress at least a portion of a pathway for 3-hydroxybutyrate utilization. Preferably, the microorganism is genetically engineered to express, enhance expression, and / or overexpress one or more genes encoding acyl-CoA synthetase and / or 3-hydroxybutyrate dehydrogenase. In some embodiments, the microorganism is engineered to express, enhance expression of, and / or overexpress (i) an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25; and / or (ii) a 3-hydroxybutyrate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:27. In some embodiments, the microorganism has been genetically engineered to introduce (i) a gene encoding an acyl-CoA synthetase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:26; and / or (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:28.

[0130] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for 3-hydroxyvalerate utilization. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and / or 3-ketoacyl-CoA thiolase. In some embodiments, the microorganism expresses (i) an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:25; (ii) an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:27; and (iii) a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:29.In some embodiments, the microorganism contains (i) a gene encoding an acyl-CoA synthetase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:26; (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:28. and (iii) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:30.

[0131] In some embodiments, the microorganism is genetically engineered to express, enhance expression, and / or overexpress at least a portion of a pathway for 1,4-butanediol utilization. Preferably, the microorganism is genetically engineered to express, enhance expression, and / or overexpress one or more genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase. In some embodiments, the microorganism has (i) a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31; (ii) a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33; or (iii) a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23. (iv) an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35; (v) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37;(vi) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5; (vii) a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:39; (viii) an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:41. and (ix) a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:43. In some embodiments, the microorganism comprises (i) a gene encoding a methanol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 32; (ii) a gene encoding a methanol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 34;(iii) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 24; (iv) a gene encoding an aldehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, or at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 36. (v) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38; (vi) a gene encoding an alcohol dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38; (vii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:6; (vii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:40; (viii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:42;and (ix) a gene encoding succinic semialdehyde dehydrogenase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:44;

[0132] In some embodiments, the microorganism is genetically engineered to express, enhance, and / or overexpress at least a portion of a pathway for the synthesis of PHA. Preferably, the microorganism is genetically engineered to express, enhance, and / or overexpress one or more genes encoding a 3-ketoacyl-CoA thiolase, an enoyl-CoA hydratase, and / or a PHA synthase. In some embodiments, the microorganism expresses (i) a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:45; (ii) a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:47; and (iii) an enoyl-CoA hydratase having at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:49; and (iv) a PHA synthase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:49.In some embodiments, the microorganism contains (i) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:46; (ii) a gene encoding an enoyl-CoA hydratase and having at least 70%, at least 75%, at least 80%, at least 85%, and (iii) a gene encoding a PHA synthase and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:50.

[0133] d. Vector In one aspect, the present invention provides a vector comprising a gene encoding an enzyme for producing polyhydroxyalkanoate (PHA) from polyester waste.

[0134] A "vector" is a tool that allows or facilitates the transfer of an object from one environment to another. According to the present invention, and by way of example, some vectors used in recombinant nucleic acid methods are capable of transferring an object, such as a segment of nucleic acid, into a target cell. A vector may serve the purpose of maintaining a heterologous nucleic acid within a cell, facilitating replication of a vector containing the nucleic acid fragment, or facilitating expression of a protein encoded by the nucleic acid fragment.

[0135] Vectors can be non-viral or viral. Examples of vectors used in recombinant nucleic acid methods include, but are not limited to, plasmids, cosmids, chromosomes, artificial chromosomes, and viruses. Vectors can be single-stranded or double-stranded. Vectors can be naked nucleic acid (e.g., DNA). Vectors used in the present invention can be, for example, naked nucleic acid, plasmids, or viral vectors.

[0136] In one embodiment, the vector is a plasmid. A "plasmid" can refer to a small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Plasmids are most commonly found in bacteria as small circular double-stranded DNA molecules, although plasmids can also exist in archaea and eukaryotes.

[0137] In one embodiment, the vector is a viral vector. Viral vectors were originally developed as an alternative to naked DNA transfection for molecular genetic experiments. Compared with traditional transfection methods, transduction efficiency can be increased, and some viruses can be integrated into the cell genome to promote stable expression. Preferably, the viral vector is a bacteriophage.

[0138] succinate pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for succinate utilization. Preferably, the vector comprises a gene encoding succinate dehydrogenase.

[0139] In one embodiment, the vector comprises a gene encoding succinate dehydrogenase. Succinate dehydrogenase (EC.1.3.5.1) is an enzyme that can catalyze the oxidation of succinate to fumarate. In one embodiment, the vector comprises a gene encoding a succinate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0140] MPKPDLPDLPDTCDLLVVGSGAAGMAAAISAHHHGLKPVIVEKSEFFGGSTAVSGGAIWVPCNPIAAAAGMTDDREAARAYIRGETGNRFNAELVDAFLDKSPEAIGFFHERTALKMAHRALSPDYHSDAPGATEGGRALDA LDYDGRRLGADLYRMRPPIADFTILGGMPLGRPDIFHFLRMTRSVKSAAYATGAVLRYFRDRLTWGRNTRLVMGAAVSGRLAETVFARNIPLFTGHELVRLLQDESGRVVGAELKGPRGVCRIAAHRGVVLAAGGYPHDAAR RAQSFEHVRRGLPHYSMSPVSGTGGGIAAAEAVGAAFVDTNPNAGFWTPVSLLRNADGSVRPFPHLFLDRAKPGVIAVGHDGRRFVNEASSYHDFVQGLIAKLLADGEKSAWLVADHRAMRRYGLGAAHAFPARIGRHVASG YLKRDATLEGLARQCGIDVATFRQTVALFNEAAARGEDPAFGKGSTSYQRYLGDGENRPNPCLRPLEGPFYAVEIYPGDIGTSMGLDITAKGEVRDSRGRTIPGLYACGNDINSVMSGAYPGPGITLGPALTFGYVIGQSAAA Exemplary succinate dehydrogenase (SEQ ID NO: 1)

[0141] Exemplary succinate dehydrogenase gene - peg.3652 (SEQ ID NO: 2)

[0142] B lactate pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for lactate utilization. Preferably, the vector comprises a gene encoding D-lactate dehydrogenase.

[0143] In one embodiment, the vector comprises a gene encoding D-lactate dehydrogenase. D-lactate dehydrogenase (EC 1.1.1.28) is an enzyme that can catalyze the conversion of lactate to pyruvate. In one embodiment, the vector comprises a gene encoding D-lactate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0144] MTRPGQAEAAADRTALLDRLRRIVGPAHVLTADRATRRYTRGFRYGEGPVAAVVRPGSLVQMWRVLNAAVASGRAVILQAANTGLTGGSTPWGQDYDREIVLVSVMRLRGIHLIGAGEQVLCLPGATLDALEKRLRPLGREP HSVIGSSCIGASVLGGICNNSGGALIQRGPAYTEMSLYAEVREDGSVALVNHLGLDLGDDPEEILARVEAGELPAPAPTDAWASDREYADHVRDIEAETPARFNADPRRLHESSGCAGKLAVFAVRLDTFQAEKDTAVFYVG SNDPDELTEIRRHILAHFQSLPIAGEYIHREAYDIAAKYGKDTFLFIRHAGTDRMPAFFAAKARMDALTERLGLGATLSDRLAQGVAALMPQHLPRRMNDFRDRYEHHLLLRMGGAGIAEARDYLGAIFPSASGAMFECTPD EGKAAFLHRFAVAGAAVRYRAIHAREVQDIVALDIALRRNDRDWVERLPPDLDAKLEKKLYYGHFFCHVFHQDYVVKKGQDCLAVEHEMWRLLDRRGAEYPAEHNVGHLYHAKPELAGFYRQLDPTNSLNPGIGQTSKCAHWH Exemplary D-lactate dehydrogenase (SEQ ID NO: 3)

[0145] Exemplary D-lactate dehydrogenase gene - peg.3013 (SEQ ID NO: 4)

[0146] C ethylene glycol pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for ethylene glycol utilization. Preferably, the vector comprises genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, and / or glyoxylate reductase.

[0147] In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase. Alcohol dehydrogenase (EC 1.1.1.1) is an enzyme that can catalyze the conversion of an alcohol to an aldehyde or ketone (e.g., converting ethylene glycol to glycolaldehyde). In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0148] MTFRANWSYPTTIKFGPGRVTELAEHCRAVGIARPLLVTDKALASLPITAQALDVLDASGLGRAVFSEVDPNPHEGNMEAGIAAYKAGGHDVICFGGSALDLGKMIALMADQTVSVWDLEDIGDWWTRADAGKIAPIIAVPTTAGTGSEVGRAGVLTNSATHKKKIIFHPRLMPAVTICDPELTVGMP KFITAGTGMDAFAHCLEAFCSPHYHPMSQGIALEGLRLVNEYLPRAYATPDDLEARAHMMSAAAMGAVAFQKGLGAIHSLSHPVGAVYGTHHGTTNAVVMMPMVLDFNRSAIEDRLARAADYLGIKGGFDGFRARVIQLRSELAIPQNLTRLGVQTERLDELTEMALEDPSCGGNPVEMTRENTRALFESCM Exemplary Alcohol Dehydrogenase (SEQ ID NO: 5)

[0149] Exemplary Alcohol Dehydrogenase Gene - peg.4723 (SEQ ID NO: 6)

[0150] In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase. Aldehyde dehydrogenase (EC 1.2.1.3) is an enzyme that can catalyze the oxidation of aldehydes (e.g., converting glycolaldehyde to glycolic acid). In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8.

[0151] MSKTIALISPATGRTLVERQTLGIEDARAAVARARAAQPEWAALPLDERIARIRAGIEALNAMKDAIVPELADQMGRPIRYGGEFGGVNERAGHMMKIAAQALAPTVVEDSDHFA REIRREPVGVVFVIAPWNYPFLTAVNTVVPALVAGNAVILKHASQTLLAGERLAEALHRGGVPAEVMQNVVLDHQTTEALIAGRSFGFVNFTGSVAGGRAIERAAAGTFTATGLEL GGKDPGYVRADADLDAAVDGLMDGAMFNSGQCCCGIERIYVHESLFDAFVAKAVDWVNAQKLGNPRDPDTTMGPMAHRRFADLVRAQVSEAVAQGARPLIDPANFPADDGGAYLA PQVLVDVTHDMRVMREESFGPVVGIMPVRDDAEAIGLMNDCDYGLTASIWTADADAAARIGSRLETGTVYMNRCDYLDPALCWTGCKDTGRGAALSGLGYLAVTRPKSYHLKKVTK Exemplary Aldehyde Dehydrogenase (SEQ ID NO: 7)

[0152] Exemplary Aldehyde Dehydrogenase Gene - peg.4722 (SEQ ID NO:8)

[0153] In one embodiment, the vector comprises a gene encoding glyoxylate reductase. Glyoxylate reductase (EC 1.1.1.79) is an enzyme that can catalyze the conversion of glycolic acid to glyoxylic acid. In one embodiment, the vector comprises a gene encoding a glyoxylate reductase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:9. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:10.

[0154] MPAVHATDPTRSRLKVTVTRRLPEAVETRMSELFDVSLNAEDRRMSREELVAAMRVSDVLVPTITDHIDAAMLAQAGDRLKLIANYGAGVDHVDVHSARQRGILVSNTPGVVTEDTADVVMALILGVTRRLPEGMAEMQAGRWQGWSPTAHLGGRLGGRRLGILGMGR IGQAVARRANAFGMQVHYHNRRRLRPEVEAELQATYWESLDQMLARMDIVSVNAPHTPSTFHLLNARRLKLLKPSAVVINTSRGEVIDENALTRMLRAGEIAGAGLDVFEHGHEINPRLRELPNVVLLPHMGSATIEGRVEMGEKVIINIKTFADGHRPPDLVVPSML Exemplary Glyoxylate Reductase (SEQ ID NO: 9)

[0155] Exemplary glyoxylate reductase gene - peg.1987 (SEQ ID NO: 10)

[0156] d adipic acid pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for adipic acid utilization. Preferably, the vector comprises genes encoding long-chain fatty acid CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxybutyryl-CoA dehydrogenase, and / or 3-oxoadipyl-CoA thiolase.

[0157] In one embodiment, the vector comprises a gene encoding a long-chain fatty acid CoA ligase. Long-chain fatty acid CoA ligase (EC 6.2.1.3) is an enzyme that can ligate acetyl-CoA to a long-chain fatty acid (e.g., adipic acid). In one embodiment, the vector comprises a gene encoding a long-chain fatty acid CoA ligase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12.

[0158] MTPKGRTGGKGMARFASVADRDAVEAEMPYAERQVPHTVYQALTETRDRHPQRPAISFQLFSDPKAPARTLTWTELHERVTETANLFRSLGVGPDDVVAYLLPNCIEAPVVLLAGATAGIVNPINPLLEPDHIAAILRETGAKVLVTLKSFPKSEVAQKA ADAVAQAPNVQTVLEVDLRGYLTGVKRLLVPLMRPKVTARHHAKVMDFDAAASAQKHNRLTFDEPAEDRVAAFFHTGGTTGMPKVAQHKQSGMIYNGWLGGTLLFTETDVLMCPLPMFHVFAAYPVLMSCLMSGAQLVMPTPAGYRGEGVFDNFWKLIER WQATFLITVPTAIAALMQRPVNADVSSLKTAISGSAPLPIELYNRFKAATGVEIAEGYGLTEATCLVSCNPINGLKKVGSVGIPLPHTHVRILQRRNGGFHECATDEIGEICVANPGVFEGSTYTEADKNHDLFAESRFLRTGDLGMDADGYLWITGRA KDLIIRGGHNIDPAEIEDALLSHPKVAAVAAIGQPDSFAGELPCAYVELIAGAEVGLDELMEHARTHIHERAAVPKHVEILPELPKTTVGKIFKPDLRKLAIRRVYDSALAEAGLAAEVGEVVDDRKRGLVAHIRPKGQVDRSAVEQLLGQYALPWEWVG Exemplary Long Chain Fatty Acid CoA Ligase (SEQ ID NO: 11)

[0159] Exemplary long chain fatty acid CoA ligase gene - peg.2056 (SEQ ID NO: 12)

[0160] In one embodiment, the vector comprises a gene encoding an acyl-CoA dehydrogenase. Acyl-CoA dehydrogenase (EC 1.3.99.3) is an enzyme that can introduce a trans double bond between C2 and C3 of an acyl-CoA thioester substrate (e.g., converting adipyl-CoA to 5-carboxy-2-pentenoyl-CoA). In one embodiment, the vector comprises a gene encoding an acyl-CoA dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14.

[0161] MSLDPETLAQFLETLDRFVRERLIPNEERVADGDAIPPELVQEIREMGLFGMSIPEEHGGIGLTMAEEVQAALVLGQASPVFRSLVGTNNGIGSQGIIIDGTPEQKAHYLPQLASGEMIASFALTEPDAGSDAGSLRCSARLDGDHYVLNGTKRFITNAPHAGLFTVFARTDPDSKSAAGVTAFLVEAGTPGL HLGPRDRKMGQKGSHTCDVILEDCRVPASAIIGGPDRLGQGFKTAMKVLDRGRLHISAVCVGAAERLIRDSLAYAMERRQFGEPIAEKQLVQAMLADSRAEAYAARCMIEETARRKDAGLSVSTEAACCKMYASEMVGRVADRAVQIHGGAGYMAEYAVERFYRDVRLFRIYEGTTQIQQLVIARNMIREASG Exemplary Acyl-CoA Dehydrogenase (SEQ ID NO: 13)

[0162] Exemplary Acyl-CoA Dehydrogenase Gene - p.200 (SEQ ID NO: 14)

[0163] In one embodiment, the vector comprises a gene encoding an enoyl-CoA hydratase. Enoyl-CoA hydratase (EC 4.2.1.17) is an enzyme that can hydrate the double bond between the second and third carbons in 2-trans / cis-enoyl-CoA. In one embodiment, the vector comprises a gene encoding an enoyl-CoA hydratase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16.

[0164] MRDISQLNLTHLLFDMDDGIATVTLNRAAKRNALNAETIEELVAVFSALPASGARAVVLRAEGPHFCAGLDLVEHGREERSPAEFMRICLRWHEAFNKIEYGGIPVIAALKGAVVGGGLELASSVHIRVMDET TYFGLPEGQRGLFTGGGATIRVPRLIGQARMMDMMLTGRLYSGDEAVQVGLAQYRVADSEAQAYDLARRVAQNTPLSNFAVCSAISHMQNMSGLDAAYAEAMVAGIVNTQDAARGRLDSFAQGTAQKIKPGEAG Exemplary Enoyl-CoA Hydratase (SEQ ID NO: 15)

[0165] atgcgcgacatttcgcagttgaacctgacccacctgctcttcgacatggacggggacggcatcgccaccgtcaccctgaaccgcgccgccaagcgcaacgccctgaatgccgagacgatcgaggaactggtcgcggtcttttccgccctgcccgcctcgggcgcccgtgccgtggtgctgcgcgccgaggggccgcatttctgcgccgggctggacctggtcgagcacgggcgcgaggaacgcagccctgccgagttcatgcgcatctgcctgcgctggcacgaggcgttcaacaagatcgaatatggcggcattccggtcatcgccgcgctcaagggcgcggtggtgggcggcgggctggaactggcctcgtcggtccatatccgggtgatggacgagaccacctatttcggcctgcccgaggggcagcgcgggctgttcaccggcggcggcgccacgatccgcgtgccccggctgatcggccaggcgcgcatgatggacatgatgctgaccggccggctgtattccggggacgaggcggtgcaggtcgggctggcgcaataccgcgtggccgacagcgaggcgcaggcctacgaccttgcccgccgggtggcgcagaacacacccctgtcgaatttcgcggtctgctcggcgatctcgcatatgcagaacatgtcggggctggacgccgcctatgccgaggccatggtcgccggcatcgtcaacacccaggacgccgccaggggaaggctggacagctttgcccagggcacggcgcaaaagatcaagccgggcgaggcaggctga Exemplary enoyl-CoA hydratase gene - peg.2628 (SEQ ID NO: 16).

[0166] In one embodiment, the vector comprises a gene encoding 3-hydroxybutyryl-CoA dehydrogenase. 3-Hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157) is an enzyme that can convert 3-hydroxybutyryl-CoA to 3-oxoadipyl-CoA. In one embodiment, the vector comprises a gene encoding 3-hydroxybutyryl-CoA dehydrogenase that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:18.

[0167] MAIQSVGVIGAGQMGNGIAHVFALAGYDVLMTDISREALDKAVAQIDHNLERQVSRGKVSAEDKAAAMRITTTTMTLSDLGKTDLIIEAATERETVKQAIFEDLLLPHLKPETILTSNTSSISITRLASRTDRPERFMGFHFMNPV PVMQLVELIRGIATNEETYKALVEVVEKIGKTSASAEDFPAFIVNRILMPMINEAVYTLYEGVGSVKSIDQSMKLGANHPMGPLELADFIGLDTCLAIMNVLHEGLADTKYRPCPLLVKYVEAGWLGRKTGRGFYDYSGEEPVPTR Exemplary 3-hydroxybutyryl-CoA dehydrogenase (SEQ ID NO: 17)

[0168] atggcgattcaatcggtgggcgtgatcggcgccggacagatgggcaatggcatcgcgcatgtctttgccctggcgggttatgacgtgctcatgaccgacatctcgcgcgaggcgctggacaaggccgtggcgcagatcgaccacaacctggaacgccaggtcagccgcggcaaggtctcggccgaggacaaggccgcggcgatgcggcgcatcaccaccaccatgacgctttccgacctgggcaagaccgacctgatcatcgaggccgccaccgagcgcgagaccgtcaagcaggcgatcttcgaggatctgctgccgcatctgaagcccgagaccatcctgacctcgaacacctcgtcgatctcgatcacccgccttgccagccgcaccgaccggcccgagcgcttcatgggcttccacttcatgaacccggttccggtcatgcagctggtcgagctgatccgcggcatcgccaccaacgaggagacctacaaagccctggtcgaagtggtcgaaaagatcggcaagacctcggccagcgccgaggatttcccggccttcatcgtcaaccgcatcctgatgccgatgatcaacgaggcggtctatacgctttacgagggcgtcggctcggtcaagtccatcgaccagtcgatgaagctgggcgccaaccacccgatggggccgctggaactggcggatttcatcggcctcgacacctgcctggcgatcatgaacgtgctgcacgaggggctggcggacacgaaataccggccctgcccgctcttggtgaaatatgtcgaggcaggctggctgggccgcaagaccgggcgtgggttctacgactattcgggcgaggagccggtgccgacgcgatag Exemplary 3-hydroxybutyryl-CoA dehydrogenase gene - peg.2620 (SEQ ID NO: 18)

[0169] In one embodiment, the vector comprises a gene encoding 3-oxoadipyl-CoA thiolase. 3-oxoadipyl-CoA thiolase (EC 2.3.1.174) is an enzyme that can convert 3-oxoadipyl-CoA to succinyl-CoA. In one embodiment, the vector comprises a gene encoding 3-oxoadipyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.

[0170] MTEVFICDYIRTPIGRFGGALSSVRADDLGAIPLRALMARHAGLDWQAVDDVIYGCANQAGEDNRNVARMSALLAGLPVEVPGTTINRLCGSGMDAVLVAARQIAAGEAELMIAGGVESMSRAPFVLPKAESAFSRHAEIHDTTIGWRFVNPAMHAAYGTDSMPQTGQNVADDYGISREAQDAMALASQQKAAAAIASG RLAAEIAPITIPQRKGEPIVVDTDEHPRATTPEALAKLRPLFPNGSVTAGNASGVNDGAAALILASEAAARKHGLTPIARVLGGATAGVPPRIMGIGPAPASQKLMDRLGLTPADFDVIELNEAFAAQGLATLRQLGIADDDPRVNPNGGAIALGPLGMSGARITGTAALELALTGGKRSLSTMCIGVGQGIAVALERV Exemplary 3-oxoadipyl-CoA thiolase (SEQ ID NO: 19)

[0171] Exemplary 3-oxoadipyl-CoA thiolase gene - peg.5206 (SEQ ID NO: 20)

[0172] e. 6-hydroxycaproic acid pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for 6-hydroxycaproic acid utilization. Preferably, the vector comprises a gene encoding an alcohol dehydrogenase and / or an aldehyde dehydrogenase.

[0173] In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase. Alcohol dehydrogenase can convert 6-hydroxycaproic acid to 6-oxocaproic acid. In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:21. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:22.

[0174] MAKTMKAAVVREFGKPLTIDEVPVPPGPGMIQVRIQASGVCHTDLHAAEGDWPVKPNPPFIPGHEGVGFVSAVGAGVKHVKEGDRVGVPWLYTACGHCRHCLGGWETLCESQLNTGYSVNGGFADYVVADPNYVGHLPKNVDFLDIAPVLCAGVTVYKGLKVTDTKPGDWV VISGIGGLGHMAVQYAKAMGMNVAAVDIDDEKLALARKLGATVTVNAATEPDPAAAIRKQTDGGAQGVLVTAVGRKAFEQAIGMVARGGTVALNGLPPGDFPLDIFGMVLNGITVRGSIVGTRLDLQESLDFAGDGKVKATVHKAKLEDINNIFGQMHKGQIEGRMVLDMAG Exemplary Alcohol Dehydrogenase (SEQ ID NO:21)

[0175] Exemplary Alcohol Dehydrogenase Gene - peg.2426 (SEQ ID NO: 22)

[0176] In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase. The aldehyde dehydrogenase can convert 6-oxocaproic acid to adipic acid. In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:23. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:24.

[0177] MPNDQTHPFRGVNALPFEERYDNFIGGEWVAPVSGRYFTNTTPITGAEIGQIARSEAGDIELALDAAHAAKEKWGATSPAERANIMLKIADRMERNLELLATAETWDNGKPIRETMAADLPLAIDHF RYFAGVLRAQEGSISQIDDDTVAYHFHEPLGVVGQIIPWNFPLLMACWKLAPAIAAGNCVVLKPAEQTPAGIMVWANLIGDLLPPGVLNIVNGFGLEAGKPLASSNRIAKIAFTGETTTGRLIMQYA SENLIPVTLELGGKSPNIFFADVAREDDDFFDKALEGFTMFALNQGEVCTCPSRVLIQESIYDKFMERAVQRVQAIKQGDPRESDTMIGAQASSEQKEKILSYLDIGKKEGAEVLTGGKAADLGGEL SGGYYIEPTIFRGNNKMRIFQEEIFGPVVSVTTFKDQAEALEIANDTLYGLGAGVWSRDANTCYRMGRGIKAGRVWTNCYHAYPAHAAFGGYKQSGIGRETHKMMLDHYQQTKNMLVSYSPKKLGFF Exemplary Aldehyde Dehydrogenase (SEQ ID NO:23)

[0178] Exemplary Aldehyde Dehydrogenase Gene - peg.2425 (SEQ ID NO: 24)

[0179] f. 3-hydroxybutyrate pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for 3-hydroxybutyrate utilization. Preferably, the vector comprises a gene encoding an acyl-CoA synthetase and / or a 3-hydroxybutyrate dehydrogenase.

[0180] In one embodiment, the vector comprises a gene encoding an acyl-CoA synthetase. An acyl-CoA synthetase is an enzyme that can catalyze the activation of free fatty acids to CoA esters (e.g., converting 3-hydroxybutyrate to 3-hydroxybutyryl-CoA and / or 3-hydroxyvalerate to 3-hydroxyvaleryl-CoA). In one embodiment, the vector comprises a gene encoding an acyl-CoA synthetase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:26.

[0181] MSATTLSRSRPGYEQAVAQFRIEDAIAGLRGDLETGLNACVECCDRHCGADRVALRCLSADEALVEYTFEDLRALSARAANLMRDKGIRPGDVVAGLLPRTVELVATVLGAWRLGAVYQPLFTAFGPKAIEHRLKTSG AKLVVTNAAQRPKLDEVEDCPLVATLRGDGPLAPGDVDFREALAMASDQFEPVMRRGEDLFMMMSTSGTTGLPKGVPVPLRALLAFGAYMRDAIDLRETDVFWNIADPGWAYGLYYALTGPLLLGQPTILYEGGFTAET TYRIIERMGVTSLAGSPTAYRLLIAAGPEAAGAVKGRLRVVSSAGEPLNPEIIRWFGEHLAVPIHDHYGQTEMGMCVNNHHGLEHPVRPGSAGLAMPGYRIVVLDDDGNELGPNQPGVLAIDMKRSPLMWFSGYLNQA TPALAGGYYRTGDSVEFEPDGSISFIGRSDDVITSSGYRIGPFDVESALIEHPAVVEAAVVGVPDPERTEIVKAFVVLAKGVEGTEALREELAQHVKKRLSAHAYPRMIDFVADLPKTPSGKIQRFVLRKAEVEKLARE Exemplary Acyl-CoA Synthetase (SEQ ID NO:25)

[0182] Exemplary Acyl-CoA Synthetase Gene - peg.2994 (SEQ ID NO: 26)

[0183] In one embodiment, the vector comprises a gene encoding 3-hydroxybutyrate dehydrogenase. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) is an enzyme that can convert 3-hydroxybutyrate to acetoacetate and / or 3-hydroxyvalerate to 3-oxopentanoate. In one embodiment, the vector comprises a gene encoding 3-hydroxybutyrate dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:27. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:28.

[0184] MFEKFLSGKTAVVTGSNSGIGLGIAHELARAGADLVLNSFTDMPEDHALAESLAAEHGVEVRYVQADMSKGADCRALIEKAGACDILVNNAGIQHVAPIPDFPGEKWDAIIAINLSSAFHTTAAALPLMR KAGWGRVINIASAHGLTASEYKSAYVAAKHGIVGLTKVTALETAKEPITCNAICPGYVLTPIVEKQIPDQMKTHNMSREDVIAKVMLQRQPSGQFATVEQMGGTAVFLCSPAAEQITGTTISVDGGWTAL Exemplary 3-hydroxybutyrate dehydrogenase (SEQ ID NO:27)

[0185] atgttcgagaaattcctcagcggcaagacggcggtggtgacgggctccaattcggggatcgggctcgggatcgcgcatgaactggcgcgcgcgggcgctgatctcgtgctgaacagctttaccgacatgcccgaggaccacgcccttgccgaaagccttgccgccgagcatggcgtcgaggtgcgctatgtccaggccgacatgtccaagggcgccgactgccgcgccctgatcgaaaaggccggcgcctgcgacatcctggtgaacaatgccggcatccagcatgtcgcaccgatcccggatttcccgggcgagaaatgggatgcgatcatcgccatcaacctgagttccgcctttcacaccacggcggcggcgctgcccctgatgcgcaaggcaggctgggggcgggtgatcaacatcgcctcggcgcacgggctgacggccagcgaatacaaatcggcctatgtcgcggccaagcacggcatcgtcggcctgaccaaggtgacggcgctggagaccgcgaaggagccgatcacctgcaacgccatctgccccggctatgtgctgaccccgatcgtggaaaagcagatccccgaccagatgaagacccacaacatgagccgcgaggacgtgatcgccaaggtcatgctgcagcgccagccctcggggcaattcgccacggtcgagcagatgggcggcacggcggtcttcctgtgctcgccggcggcggagcagatcaccggcacgaccatctcggtggacggggggtggacggcgctttag Exemplary 3-hydroxybutyrate dehydrogenase gene - peg.960 (SEQ ID NO: 28)

[0186] g. 3-Hydroxyvalerate pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for 3-hydroxyvalerate utilization. Preferably, the vector comprises genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and / or 3-ketoacyl-CoA thiolase.

[0187] In one embodiment, the vector comprises a gene encoding 3-ketoacyl-CoA thiolase. 3-Ketoacyl-CoA thiolase (EC 2.3.1.16) is an enzyme that can decompose 3-oxopentanoyl-CoA into acetyl-CoA and propionyl-CoA. In one embodiment, the vector comprises a gene encoding a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:29. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:30.

[0188] MSENEIVILSGARTAIGTFGGSLAGVPPIQLAATVTRAAIERAGIGPERIGTVVFGHVLNTEPRDMYLSRVAMLDAGVPDTTPAMNVNRLCGSGAQAIVSATQALILGDADFAVAGGAESMSRAPYAVPAARFGAKMGDVQMLDMMVGALTCPMGTGHMGVTAENVAREHDISRQAQDEFALESQKRAAAAIAEGR FKEQIVPIEIKTRKGMVAFDTDEHPKATDLEKLAGLKAVFQKDGTVTAGNASGINDGAAALVLARADAARAAGAKPLFRVLGYAVAGVRPEVMGIGPVPAVEALLKSTGLKIGEFDVIESNEAFAAQALAVNKGLGLDPAKVNPNGGAIALGHPVGATGALVTVKAMYELMRTGGSKGLITMCIGGGQGIALAIERI Exemplary 3-ketoacyl-CoA thiolase (SEQ ID NO:29)

[0189] Exemplary 3-ketoacyl-CoA thiolase gene - peg.2743 (SEQ ID NO: 30)

[0190] h. 1,4-butanediol pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for 1,4-butanediol utilization. Preferably, the vector comprises genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase.

[0191] In one embodiment, the vector comprises a gene encoding methanol dehydrogenase. Methanol dehydrogenase (EC 1.1.2.7) is an enzyme that can convert 1,4-butanediol to 4-hydroxybutyraldehyde. In one embodiment, the vector comprises a gene encoding a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.

[0192] MKNLMNGACLALLMSGTAALANDSVLAEIAKQQWAIQMGDYANTRYSTLDQINKDNVKDLRVAWTFSTGVLRGHEGSPLVIGDVMYVHTPFPNRVFALDLNDNGKILWRYEPQQDPNVIAVMCCDTVYRGLSYADGMILLGQADTTVVA LDATSGEVKWSTKIGDPGIGETLTATVVPVKDKVLVGISGGEYGVRGRMTALNLTDGSEAWKAWSTGPDEELLVDPETTTTHLGKPIGADSSLNSWEGDQWQIGGGTIWGWFSYDPDLNLVYYGTGNPSTWNPSQRPGDNKWSMTIMARDA DTGMAKWFYQMTPHDEWDYDGVNEMILTNQTVDGQERKLLTHFDRNGLAYTMDRETGELLVAEKYDPVVNWTTGVDMDPNSETYGRPAVVAEYSTAQNGEDENTTGVCPAALGTKDQQPAAFSPKTNLFYVPTNHVCMDYEPFRVAYTAG QPYVGATLSMYPAPNSHGGMGNFIAWDNTTGEIKWSVPEQFSVWSGALATAGDVVFYGTLEGYLKAVDAQTGEELYKFKTPSGIIGNVMTYEHGGKQYVGILSGVGGWAGIGLAAGLTNPNDGLGAVGGYASLSQYTELGGQLTVFELPG Exemplary Methanol Dehydrogenase (SEQ ID NO: 31)

[0193] Exemplary Methanol Dehydrogenase Gene - peg.20 (SEQ ID NO: 32)

[0194] In one embodiment, the vector comprises a gene encoding a methanol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0195] MNRNTPKARGASSLAMAVMGLAVLTTAPATANDQLVELAKDPANWVMTGRDYNAQNYSEMTDINKENVKQLRPAWSFSTGVLHGHEGTPLVVGDRMFIHTPFPNTTFALDLNEPGKILWQNKPKQNPTARTVACCDVVNRGLAYWPGDDQVKPLIFR TQLDGHIVAMDAETGETRWIMENSDIKVGSTLTIAPYVIKDLVLVGSSGAELGVRGYVTAYDVKSGEMRWRAFATGPDEELLLAEDFNAPNPHYGQKNLGLETWEGDAWKIGGGTNWGWYAYDPEVDLFYYGSGNPAPWNETMRPGDNKWTMAIWGRE ATTGEAKFAYQKTPHDEWDYAGVNVMMLSEQEDKQGQMRKLLTHHPDRNGIVYTLDRTNGDLISADKMDDTVNWVKEVQLDTGLPVRDPEFGTRMDHKARDICPSAMGYHNQGHDSYDPERKVFMLGINHICMDWEPFMLPYRAGQFFVGATLTMYPGP KGDRQNALGLGQIKAYDAISGEMKWEKMERFSVWGGTMATAGGLTFYGTLDGFIKARDSDTGDLLWKFKLPSGVIGHPMTYKHDGRQYVAIMYGVGGWPGVGLVFDLADPTAGLGSVGAFKRLQEFTQMGGGVMVFSLDGESPYSDPNVGEYAPGEPT Exemplary Methanol Dehydrogenase (SEQ ID NO: 33)

[0196] Exemplary Methanol Dehydrogenase Gene - peg.3083 (SEQ ID NO: 34)

[0197] The aldehyde dehydrogenase can convert 4-hydroxybutyraldehyde to 4-hydroxybutyric acid. In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36.

[0198] MTRSFDPDTLDLPRGHFIAGEHVADRGRLAMHRPSDGAAFGESPVADADMVDRAVAAGRAALAASGWGCGVPRDRTRALLKWADLIEAEAETLARFEAACSTRPVAQLPVGDIAVTAEQIR FFAEMADKEGSDLVPTRDASLGMTVDEPYGVVGAITPWNFPLSMAGWKLAPALAAGNAVVLKPSEMTPFSTLYMAELSVRAGIPAGLVNVVLGDGPVTGNAITGHPGIGKVSFTGSTGAGQA IMGNIARNGVKPMTLELGGKSPQIVFADADLDLAADCIARSITFNAGQACVAGSRVLVAAEIIAEALAERLIARMADHRPGTTWDAETQYSPIISERQIARIDGIVQAAVAQGAEVLAGAARL DHPGWFYAPTLLAGVAPDSPAVTEEIFGPVLTLEPFADEEQAVAMADHPTYGLCAGIFTRDLSCALRVMRRIEAGTVWINRYGRSRDHILPTGGYKSSGIGKDLGRAAYHANRRQKSVLIDL Exemplary Aldehyde Dehydrogenase (SEQ ID NO: 35)

[0199] Exemplary Aldehyde Dehydrogenase Gene - peg.5153 (SEQ ID NO: 36)

[0200] The alcohol dehydrogenase can convert 4-hydroxybutyrate to succinic semialdehyde. In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0201] MDFTNLGVMRAPRHLVFGAGQRGALARHAGVFGTRALIVTDTRMARDKDFLQMRQALEAQGIATQVFDGVAAELPLSCIEAGAKAGRAAGARMIIGIGGGSCLDAAKIIGLLLSHGGAPQDYYGEYKVPGPIMPLILLPTTSGTGSEVTPVAVLDDPQRAMKIGIASPHLIPEIAICDPELTLSCPPGLTAASGADAMTHAIEAFTTLR RPADSGLSLDHVFIGKNAISDSLALEAIRLIAANLARCVSHGDDLEARSAMMLGSTLAGLAFGVAGTAAAHAIQYPVGAMTHTAHGLGVATLMPYVMAWNRPSCETDFARIGAAMGLAASGDTSRQAEAAIAAIAALFAQVGIPATIAQLGVPEDRLDEIARLALSAERLIKNNPRMLDAEGMDRIVRAAHSGDLDLLTATSPRKAALQ Exemplary Alcohol Dehydrogenase (SEQ ID NO: 37)

[0202] Exemplary Alcohol Dehydrogenase Gene - peg.245 (SEQ ID NO: 38)

[0203] In one embodiment, the vector comprises a gene encoding succinic semialdehyde dehydrogenase. Succinic semialdehyde dehydrogenase (EC 1.2.1.16) is an enzyme that can catalyze the oxidation of succinic semialdehyde to succinic acid. In one embodiment, the vector comprises a gene encoding a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0204] MSYHDTISQELGFDPQALRGDLWIDGGWRKGRGGDPIAVIDPSTGNTITRIENASIDDAMDAVAAAEAALPGWAATPPRVKSEILRRCYDLMIQRKDMLARLISLENGKALPDAQGEVLYAAE FFRWFAEEAVRLNGEIYTAPSGANRIIVTHRPIGVAVMVTPWNFPAAMATRKIAPALAAGCTCVLKPATETPLTAYALAEIYAEAGVPPGVVNVLTTSRSGATVSAMLHDPRVRKLSFTGSTE VGRRLLHEAADTVISCSMELGGNAPFIVFDDADLDLAIEGAMVAKMRNGGEACTAANRFLVQKGIAPAFAERLAARMEAMTLGAGYAGETLCGPLINREALDRIAGLVSEAESHGAKTLTGGR PLDRPGFYFPPTVLTDVPPQAEITGEEIFGPVAALATFETEDEAIARANSTEYGLISYVFTSDLARGLRVSERLDSGMVGLNRGVVSDPAAPFGGTKQSGLGREGAHHGILEFCEVQYIAANW Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO: 39)

[0205] Exemplary succinic semialdehyde dehydrogenase gene peg.246 (SEQ ID NO: 40)

[0206] In one embodiment, the vector comprises a gene encoding a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42.

[0207] MTKHATDLKMLLKDPSLLQTRAYVAGEWVDADDGKTFPVVNPARGDVIAEVADLSRAEVARAIAAAAEAMKGWAARTAKGRAQIMRKWFDLMMENQDDLGRILTAEMGKPLPEAKGEIAYGA SFIEWFGEEAKRIYGETIPGHLPDKRLTVIRQPIGVVGSITPWNFPNAMITRKCGPAIAAGCGFVGRPAAETPLSALALAVLAERAGIPKGLFSIVTSSRSSDIGKEFCENPLIRKLTFTGST EVGRILLRQAADQVLKCSMELGGNAPFIVFDDADLDAAVEGAMASKFRNNGQTCVCANRIYVQAGVYDAFAQKLAAAVDKLRVGDGLEEGVTTGPLINQDAVEKVQEHIQDAVAGGATVVTG GKPREGLFFDPTVVTGITDKMKVATEETFGPLAPLFETEEEAVERANATIFGLASYFYARDIGRITRVQEALEYGIVGVNTGIISTEVAPFGGVKQSGLGREGSRHGIEDYLEMKYICLSI Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO: 41)

[0208] Exemplary succinic semialdehyde dehydrogenase gene - peg.2316 (SEQ ID NO: 42)

[0209] In one embodiment, the vector comprises a gene encoding a succinic semialdehyde dehydrogenase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44.

[0210] MTALKDKDLLRQQALIGGNWVDAASGAVVQVTDPATGQVMGTIPDLSAAETRAAIDAADAAFASWKKRSHAERAALLERWFDLMNQHAEDLALILTLEQGKPLSEARGEIAYGASFVKWF AEEARRIDGTVIPAPTNDRRILTLKEPVGVSAIITPWNFPNAMITRKVGPALAAGCTVVIKPSEFTPYSALALGVLAERAGIPAGVVNIVTGMPAEIGAELTANPTVRKVSFTGSTRVGSL LMAQSAPTVKRLSLELGGNAPFIVFDDADLDAAVEGAIASKFRNGGQTCVCSNRILVQAGVYDAFAEKLGAKVAAMKVGPGTQAGNDIGPMINRAALDKIARHVADAVAKGATVAARAEI PEGQYAAPVVLTGATTEMELASEETFGPVAPLFRFETEDEAVAIANGTPFGLAAYFYTENIRRAWRVAEALEFGMVGLNTGAVSTTVSPFGGVKSSGLGREGARAGIEEYLEVKAFHMGGL Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO: 43)

[0211] Exemplary succinic semialdehyde dehydrogenase gene - peg.2992 (SEQ ID NO: 44)

[0212] i. PHA synthesis pathway In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for the synthesis of PHA. Preferably, the vector comprises genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and / or PHA synthase.

[0213] 3-ketoacyl-CoA thiolase can catalyze the condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. In one embodiment, the vector comprises a gene encoding a 3-ketoacyl-CoA thiolase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46.

[0214] MSTDPIVIVGSARTPMGGFQGDFAGVEAAALGATAIKAALGGLDPQAVDEIIMGCVLPAGQGQAPARQAALGAGLPLGAGATTVNKMCGSGMKAAMLGHDLILAGSADVVVAGGMESMSNAPYLLPKARSGYRMGHGQVMDHMFLDGLEDAYDKGRLMGTFAEDCAEAYQFTREAQDEFAISSLTRAQKAIAAGHF TGEIAPVTVRGRGGETVVDTDEQPGKARPDKIPTLRPAFRKDGTVTAANSSSISDGAAALVLMRASEAERRGLVPRARILGHATFADKPGLFPTAPIGSVRRLLERTGTAIGDYDLFEVNEAFAVVAMAAMRDLGLSHDAVNVHGGACALGHPIGASGARVLVTLLAALETHGGRRGIASLCIGGGEATAVAIERMQ Exemplary 3-ketoacyl-CoA thiolase (SEQ ID NO: 45)

[0215] Exemplary 3-ketoacyl-CoA thiolase gene - peg.2992 (SEQ ID NO:46)

[0216] In one embodiment, the vector comprises a gene encoding an enoyl-CoA hydratase. Enoyl-CoA hydratase (EC 4.2.1.17) may also function as a delta(3)-cis-delta(2)-trans-enoyl-CoA isomerase (EC 5.3.3.8), a 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and / or a 3-hydroxybutyryl-CoA epimerase (EC 5.1.2.3) to synthesize hydroxybutyryl-CoA. In one embodiment, the vector comprises a gene encoding an enoyl-CoA hydratase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:47. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:48.

[0217] MPVHYDLAGDSAVLTFDNPPLNVLGQAMRADLARAIAQAAADRPARLILRGAGRNFVAGADAREFDGPPLDPQLNEVLDALAALPFPTIAAIHGAALGGGLEIALACRFRIAHPSATLGLPEVTLGIVPGAGGTQRLPRLVGMAAALDLLGQGRSVTAAEAESLGLIDLIAD DPMAAARGVDTQTLLRALCADDRPPPAPDEAAVAAAHARADRRAPGQVAPHRAIELVATSAQEPIKAALTRERATFLDLRGSDQARALRHVFFAERAAMAQGKAWPAPEIARAVVVGGGNMGAAIAYALLSAGLVVRVVETDAAALDRARDNIAGLVAQGRKRGALTDAGA AELQARLSLAVGYDDLPAADLAIEAAYEDMAVKQAIFAALQDALPDSTILATNTSYLDIDLLAQGIRQPGRFLGLHFFAPAHVMKLLEIVRGEATSDQTLGAAFRLARKLGKVPVLAGVCDGFIGNRILARYRHAADILLLEGALPAQVDAAMRGFGMAMGPYEAQDMSGLD IAYANRRRQNLRDRADHRYVPIADHLVERCRRLGRKSGAGWYDYDAEGRAQPSDEVTQAILSASRDAGITRVALPAEGIAERLVLAMIAEATRILAEGIAAAPRDIDLVLVHGYGFPRWRGGGLMHHADRLTPARILSRIEALAKDDPLSWSVPPLLRQLADEGRDFTSLNPSA Exemplary Enoyl-CoA Hydratase (SEQ ID NO: 47)

[0218] Exemplary enoyl-CoA hydratase gene - peg.203 (SEQ ID NO: 48)

[0219] In one embodiment, the vector comprises a gene encoding a PHA synthase. In one embodiment, the vector comprises a gene encoding a PHA synthase having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49. In one embodiment, the vector comprises a gene comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50.

[0220] MAGKDKKPEVEAGAGDAAKPRKARGTGAKARSAAKVAKQDPAPADSEAPARGRRAKAAPAAKAAQEPVTLAKPKAAAGSGAGKPAAEPVAKSGARKPVSRARAASRSDPRTGTRRKPSAKAAAKAQAEAASLSAVDEALRPLGAVGPGAAPPMAAPAAASPTPAGTERPAAPAAEPSSAAAFAEAAFGIGSRL PEQLAQNIERIESLTQRLISALAQRRPHSPGVELPGPELFATATGAWIKLLTEQPERVLSQQVSYWGETLRHFAEAQAALARGTLKPPPSEGLRDRRFSNPLWEAHPFFNFIKRQYQINAQALEEAASALDLPEMTDRRRIEWFTRQMIDMMAPTNFLATNPDALEKALETEGESLVKGLENLVRDVEQNNGE LIVSLADRDAFRVGENIGTTEGTVVARTKLYELIQYKPTTAQVHEIPLVIFPPWINKFYILDLKPQNSLIKWIVDQGYTLFVVAWKNPDPSYGDTGMDDYVTAYLEVMDRVLDLTDQKKLNVVGYCIAGTTLALTLSILKQRGDDRVNSATFFTALTDFADQGEFTAYLQEDFVSGIEEEAARTGVLGAQLMT RTFSFLRANDLVWGPAIRSYMLGEMPPAFDLLFWNGDGTNLPGRMAVEYLRGLCQQNRFVKEGFDLLGHRLHVGDVTVPLCAIACETDHIAPWRDSWRGVAQMGSKDKTFILSESGHIAGIVNPPSKKKYGHYTSDAGFDQGEQHWLDKARHHEGSWWGRWGEWLARRAGNMVEARDPGEGFGPAPGLYVHERA Exemplary PHA Synthase (SEQ ID NO: 49)

[0221] Exemplary PHA synthase gene - peg.988 (SEQ ID NO:50)

[0222] e. Cells and kits In one aspect, the present invention provides a cell comprising a vector according to the present invention. The cell may be an isolated cell.

[0223] The cell may be a microorganism, such as a bacterium, archaea, fungus, or protist. In some embodiments, the cell is a bacterium. In some embodiments, the bacterium is from the family Rhodobacteraceae. In some embodiments, the bacterium is from the genus Paracoccus. In some embodiments, the bacterium is Paracoccus denitrificans, Paracoccus pantotrophus, or Paracoccus verustus. In some embodiments, the bacterium is Paracoccus denitrificans.

[0224] In one aspect, the present invention provides a kit for producing polyhydroxyalkanoates (PHAs) from polyester waste.

[0225] The kit may include one or more microorganisms according to the invention. The kit may include one or more vectors according to the invention. The kit may include instructions for carrying out the methods of the invention.

[0226] [Example] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way.

[0227] f. Example 1: Creation of a Genome-Scale Model (GSM) We have identified genetic traits that enable the metabolic conversion of various biodegradable polyester wastes and their monomers to form novel biologically synthesized polyesters (PHAs) by the same microorganism (e.g., Paracoccus denitrificans).

[0228] Genetic traits were identified by de novo generation of a genome-scale model (GSM) based on the published genome of P. denitrificans PD1222 (see GenBank assembly accession number: GCA_000203895.1). P. denitrificans PD1222 is a derivative of P. denitrificans DSM 413. The GSM was used for metabolic reconstruction by identifying genes previously reported in different microorganisms for the catabolism of each polyester component, as well as genes identified to produce the PHA polyester PHB, in the genome. Using the GSM, we were able to not only identify the genes present in the genome but also to understand the formation of possible metabolic networks in the organism, thus identifying plausible relationships between the activities of different genes and metabolic outcomes. The targeted components were most of the monomers used in the industrial production of major plastic polyesters.

[0229] Table 1 shows the genetic traits and network connections identified in the genome related to the utilization of a vast number of polyester monomers via aerobic or anaerobic metabolic pathways in Paracoccus denitrificans. Table 1 also shows the genetic traits and network connections identified related to the production of PHA (in the form of PHB) by Paracoccus denitrificans. Figure 2 shows a visualization of the metabolic potential for the conversion of polyester monomers by Paracoccus denitrificans based on a newly constructed genome-scale model (GSM).

[0230] The present inventors have discovered for the first time that this microorganism has a pathway for 1,4-butanediol. In particular, 1,4-butanediol is one of the constituent monomers of various polyesters and is toxic to humans, making it highly suitable for achieving biological degradation and recycling.

[0231] In the examples below, a derivative of P. denitrificans DSM 413 was used, based on a newly constructed GSM.

[0232] Table 1. Summary of identified genes that, when present in the genome, enable metabolic utilization of different polyester monomer components and subsequent production of biopolyesters (PHB). These genes are part of the metabolic pathways identified in P. denitrificans PD1222 and are also present in other P. denitrificans strains, including P. denitrificans ATCC 19367 (see Si, YY, et al., 2019, Canadian Journal of Microbiology, 65(7), pp. 486-495), P. denitrificans DYTN-1 (see Zhao, Y., et al., 2020, Letters in Applied Microbiology, 70(4), pp. 263-273), and P. denitrificans R-1 (see Hu, C., et al., 2022, Microbiology Resource Announcements, 11(4), pp. e01236-21).

[0233] [Table 1] TIFF2025535284000004.tif180147

[0234] g. Example 2: In vivo metabolism of polyester monomers and production of PHA under aerobic conditions Each polyester monomer was tested separately in minimal medium as the sole carbon source to determine whether P. denitrificans could grow on, and therefore metabolize, this substrate, and to determine which monomer was consumed more rapidly by P. denitrificans.

[0235] To test the growth ability on the substrate as the sole carbon source, batch cultures were performed aerobically at 34°C in mineral salts medium (MSM) and the corresponding monomer as the sole carbon source. A 2% inoculum was obtained from a culture in LB medium under the same conditions. The results in Table 2 confirm that P. denitrificans can degrade the monomers of 12 different plastic polymers used, for example, in packaging materials.

[0236] Kinetic analysis was obtained from batch cultures carried out in 2 L fermentors aerobically at 37°C in mineral salts medium (MSM) and the corresponding monomers (0.3% w / v) as the sole carbon source. The inoculum was grown at an OD of approximately 0.1. 600 The maximum growth (maximum OD) was measured by the same conditions as in Example 1. 600 The culture was continued until the α- and β-actin concentrations reached 0.3% (weight / volume) of the medium. The data presented are triplicate results. Figure 3 shows the biomass formation (cell mass dry weight) of P. denitrificans when different monomers were supplied as the sole carbon source at 0.3% (weight / volume) of the medium. Table 3 shows that high yields can be obtained even with polyester monomers that are generally difficult to process using microorganisms, such as 1,4 butanediol.

[0237] [Table 2]

[0238] [Table 3]

[0239] Analysis of the resulting microbial broth by GC-MS confirmed the ability of P. denitrificans to produce PHA polymers from a wide variety of polyester monomers as substrates. Figure 4 and Table 4 show that, depending on the monomer used as the substrate, P. denitrificans produces PHA polymers with different ratios of the different components of the PHA polymer. The ratios of the PHA species also vary.

[0240] [Table 4]

[0241] h. Example 3: In vivo metabolism of polyester monomers and production of PHA under anaerobic conditions Batch cultures were performed anaerobically at 34°C in mineral salts medium (MSM) and succinic acid (the monomer of PBS, PBSA, PBST, PBSTIL) as the sole carbon source. A 2% inoculum was obtained from a culture in LB medium under the same conditions. Figure 5 and Table 5 demonstrate that P. denitrificans was able to utilize polyester monomers to produce PHA under anaerobic conditions.

[0242] [Table 5]

[0243] i. Example 4: In vivo metabolism of polyester polymer and production of PHA Biological polymer recycling by P. denitrificans can be achieved not only for single monomers but also for polymers. In waste management, polymers (e.g., from packaging materials) arrive intact at waste treatment centers. Mechanical (e.g., shredding) and / or chemical (e.g., alkaline) treatments promote the breakdown of complex compounds into smaller particles, making the polymer monomers available for recycling. Biorecycling by P. denitrificans can also be achieved from pretreated polymers, resulting in the production of new biopolymers (e.g., PHAs).

[0244] The polymers were shredded (500-1000 μm) and chemically treated with alkaline solution (2 M NaOH) and incubated at 37 °C for 7 days under constant agitation (300 rpm). The polymers tested were PHB, PLA, PHBV, PHBH, and PLA / PCL. The alkaline-pretreated polymers were neutralized (to pH 7 with HCl) and added to mineral salts medium (90% v / v) as the sole carbon source at 10% v / v. Culture bottles were inoculated with 1 mL of fresh P. denitrificans culture and incubated at 30 °C for 3 days under orbital shaking at 300 rpm. Biomass growth (estimated as optical density) and PHB content were monitored periodically.

[0245] Figure 6 shows that bacteria grew and produced PHB on all pretreated polymers as the sole carbon source. When PHB, PHBH, or PHBV hydrolysates were used as substrates, PHB accumulated to a maximum of 30% of the microbial cell mass. When PLA and PCL / PLA blends were used, the PHB accumulation was 15%. When PBS and PBAT hydrolysates were used, the accumulation was approximately 6%.

[0246] Embodiment Various preferred features and embodiments of the present invention are described with reference to the following numbered paragraphs.

[0247] 1. A method for producing polyhydroxyalkanoate (PHA) from polyester waste, comprising: (a) providing a culture broth containing polyester waste; and (b) culturing a microorganism in the culture broth to produce PHA, wherein the microorganism produces the PHA by utilizing multiple polyester monomers from the polyester waste.

[0248] 2. A method for producing polyhydroxyalkanoate (PHA) from polyester waste, comprising: (a) preparing a culture broth containing polyester waste; and (b) culturing a microorganism in the culture broth to produce PHA, wherein the microorganism utilizes 1,4-butanediol from the polyester waste to produce the PHA.

[0249] 3. The method of paragraph 1 or 2, wherein the microorganism is from the genus Paracoccus.

[0250] 4. The method of any one of paragraphs 1 to 3, wherein the microorganism is Paracoccus denitrificans.

[0251] 5. The method of any one of paragraphs 1 to 4, wherein the microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof.

[0252] 6. The microorganism is Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, or Paracoccus denitrificans NBRC 102528, Paracoccus denitrificans NCCB 22021, Paracoccus denitrificans NBRC 13301, Paracoccus denitrificans NCIMB 8944, Paracoccus denitrificans DSM 15418, Paracoccus denitrificans DSM 415, Paracoccus denitrificans NCIMB 11627, Paracoccus denitrificans NCIMB 9722, Paracoccus denitrificans IMET 10380, Paracoccus denitrificans VKM B-1324, or Paracoccus denitrificans ICPB 3979.

[0253] 7. The method of any one of paragraphs 1-6, wherein the microorganism comprises genes encoding two or more pathways, three or more pathways, four or more pathways, five or more pathways, six or more pathways, or seven or more pathways selected from the following: (i) a pathway for succinate utilization; (ii) a pathway for lactic acid utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0254] 8. The method of any one of paragraphs 1 to 7, wherein the microorganism comprises a plurality of genes encoding each of the following pathways: (i) a pathway for succinate utilization; (ii) a pathway for lactic acid utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0255] 9. The method of any one of paragraphs 1 to 8, wherein the polyester waste comprises 1,4-butanediol.

[0256] 10. The method of any one of paragraphs 1 to 9, wherein the polyester waste comprises two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

[0257] 11. The method of any one of paragraphs 1 to 10, wherein the polyester waste comprises succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

[0258] 12. The method of any one of paragraphs 1 to 11, wherein the polyester waste comprises the polyester monomer in the form of free monomer.

[0259] 13. The method of any one of paragraphs 1 to 12, wherein the polyester waste comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more polyesters selected from polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), poly(ethylene adipate) (PEA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0260] 14. The method of any one of paragraphs 1 to 13, wherein the polyester waste is pretreated, and optionally the polyester waste is mechanically treated and / or chemically treated.

[0261] 15. The method of any one of paragraphs 1 to 14, further comprising pretreating the polyester waste.

[0262] 16. The method of any one of paragraphs 1 to 15, further comprising mechanically treating the polyester waste.

[0263] 17. The method of paragraph 16, wherein the polyester waste is shredded, and optionally the polyester waste is shredded to a particle size of about 100 μm to about 5000 μm, about 200 μm to about 4000 μm, about 300 μm to about 3000 μm, about 400 μm to about 2000 μm, or about 500 μm to about 1000 μm.

[0264] 18. The method of any one of paragraphs 1 to 17, further comprising chemically treating the polyester waste.

[0265] 19. The method of any one of paragraphs 1 to 8, wherein the polyester waste is subjected to an alkaline treatment.

[0266] 20. The method of paragraph 19, wherein the polyester waste is neutralized after the alkaline treatment.

[0267] 21. The method of any one of paragraphs 1 to 20, wherein the culture broth comprises the polyester waste in an amount of about 1 g / L to about 100 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 20 g / L, about 2 g / L to about 10 g / L, or about 2 g / L to about 5 g / L.

[0268] 22. The method of any one of paragraphs 1 to 21, wherein the culture broth comprises a mineral salts medium.

[0269] 23. The method of any one of paragraphs 1 to 22, wherein the microorganism is cultured under aerobic or anaerobic conditions.

[0270] 24. The method of any one of paragraphs 1 to 23, wherein the microorganism is cultured under anaerobic conditions.

[0271] 25. The method of any one of paragraphs 1 to 24, wherein the microorganism is cultured for about 1 day to about 7 days, about 2 days to about 6 days, or about 3 days to about 5 days.

[0272] 26. The method of any one of paragraphs 1 to 25, wherein a single microbial strain is cultured.

[0273] 27. The method of any one of paragraphs 1 to 26, comprising a single culture step.

[0274] 28. The method of any one of paragraphs 1 to 27, wherein the microorganism utilizes at least three, at least four, at least five, at least six, at least seven, or at least eight polyester monomers from the polyester waste to produce the PHA.

[0275] 29. The method of any one of paragraphs 1 to 28, wherein the microorganism utilizes polyester monomers from a plurality of polyesters from the polyester waste to produce the PHA, and optionally, the microorganism utilizes at least three, at least four, at least five, at least six, at least seven, or at least eight polyester monomers from the polyester waste to produce the PHA.

[0276] 30. The method of any one of paragraphs 1-29, wherein at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 75% by weight, or at least about 80% by weight of the polyester waste is utilized during the culturing.

[0277] 31. The method of any one of paragraphs 1 to 30, wherein at least about 0.01 mg / mL, at least about 0.02 mg / mL, at least about 0.03 mg / mL, at least about 0.04 mg / mL, at least about 0.05 mg / mL, or at least about 0.1 mg / mL of PHA is produced.

[0278] 32. The method of any one of paragraphs 1-31, wherein at least about 10 μg PHA / mg dry cell weight (DCW), at least about 20 μg PHA / mg DCW, at least about 30 μg PHA / mg DCW, at least about 40 μg PHA / mg DCW, or at least about 50 μg PHA / mg DCW is produced.

[0279] 33. The method of any one of paragraphs 1 to 32, wherein the PHA comprises or consists of polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof.

[0280] 34. The method of any one of paragraphs 1 to 33, wherein the PHA comprises or consists of polyhydroxybutyrate (PHB) or a copolymer thereof.

[0281] 35. The method of any one of paragraphs 1 to 34, wherein the PHA comprises or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0282] 36. The method of any one of paragraphs 1 to 35, further comprising recovering the PHA.

[0283] 37. A culture broth comprising polyester waste and a microorganism, wherein the microorganism is capable of utilizing a plurality of polyester monomers from the polyester waste to produce a PHA.

[0284] 38. The culture broth according to paragraph 37, wherein the microorganism is as defined in any one of paragraphs 3 to 8, the polyester waste is as defined in any one of paragraphs 9 to 14, and / or the culture broth is as defined in paragraph 21 or 22.

[0285] 39. The culture broth of paragraph 37 or 38, further comprising a PHA, optionally wherein the PHA is as defined in any one of paragraphs 33 to 35.

[0286] 40. A polyhydroxyalkanoate (PHA) produced by the method of any one of paragraphs 1 to 39.

[0287] 41. An article comprising or consisting of a PHA according to paragraph 40.

[0288] 42. An article according to paragraph 41 that is packaging material.

[0289] 43. Use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism utilizes multiple polyester monomers from the polyester waste to produce PHA.

[0290] 44. Use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism utilizes 1,4-butanediol from the polyester waste to produce PHA.

[0291] 45. The use according to paragraph 44 or 45, wherein the microorganism is as defined in any one of paragraphs 3 to 8 and / or the polyester waste is as defined in any one of paragraphs 9 to 14.

[0292] 46. ​​The use of any one of paragraphs 43 to 45, wherein the microorganism utilizes at least three, at least four, at least five, at least six, at least seven, or at least eight polyester monomers from the polyester waste to produce PHA.

[0293] 47. The use of any one of paragraphs 43 to 46, wherein the microorganism utilizes polyester monomers from a plurality of polyesters from the polyester waste to produce a pre-PHA, and optionally the microorganism utilizes at least three, at least four, at least five, at least six, at least seven, or at least eight polyester monomers from the polyester waste to produce the PHA.

[0294] 48. The use according to any one of paragraphs 43 to 47, wherein the PHA comprises polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof.

[0295] 49. The use according to any one of paragraphs 43 to 48, wherein the PHA comprises or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0296] 50. The use according to any one of paragraphs 43 to 49, wherein the microorganism produces the PHA under anaerobic conditions.

[0297] 51. The use according to any one of paragraphs 43 to 50, wherein the microorganism produces the PHA in a single cultivation step.

[0298] 52. A microorganism for producing polyhydroxyalkanoates (PHAs) from polyester waste, the microorganism comprising genes encoding pathways for the utilization of multiple polyester monomers and genes encoding pathways for the synthesis of PHAs, and which has been genetically engineered to express at least a portion of one or more of the pathways.

[0299] 53. The microorganism of paragraph 52, comprising genes encoding one or more pathways, two or more pathways, three or more pathways, four or more pathways, five or more pathways, six or more pathways, or seven or more pathways selected from: (i) a pathway for succinate utilization; (ii) a pathway for lactic acid utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0300] 54. The microorganism of paragraph 52 or 53, comprising genes encoding (i) a pathway for succinate utilization; (ii) a pathway for lactate utilization; (iii) a pathway for ethylene glycol utilization; (iv) a pathway for adipic acid utilization; (v) a pathway for 6-hydroxycaproic acid utilization; (vi) a pathway for 3-hydroxybutyric acid utilization; (vii) a pathway for 3-hydroxyvaleric acid utilization; and (viii) a pathway for 1,4-butanediol utilization.

[0301] 55. A microorganism according to any one of paragraphs 52 to 54, which has been genetically engineered to express at least a portion of a pathway for succinate utilization, and optionally, one or more genes encoding succinate dehydrogenase.

[0302] 56. The microorganism of any one of paragraphs 52 to 55, which has been genetically engineered to express a succinate dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0303] 57. The microorganism of any one of paragraphs 52 to 56, which has been genetically engineered to introduce a gene encoding succinate dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 2.

[0304] 58. A microorganism described in any one of paragraphs 52 to 57, which has been genetically engineered to express at least part of a pathway for lactate utilization, and optionally, one or more genes encoding D-lactate dehydrogenase.

[0305] 59. The microorganism of any one of paragraphs 52 to 58, which has been genetically engineered to express a D-lactate dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0306] 60. A microorganism according to any one of paragraphs 52 to 59, which has been genetically engineered to introduce a gene encoding D-lactate dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 4.

[0307] 61. The microorganism of any one of paragraphs 52 to 60, which has been genetically engineered to express at least a portion of a pathway for ethylene glycol utilization, and optionally, one or more genes encoding an alcohol dehydrogenase, an aldehyde dehydrogenase, and / or a glyoxylate reductase.

[0308] 62. The microorganism of any one of paragraphs 52 to 61, which has been genetically engineered to express one or more of: (i) an alcohol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 5; (ii) an aldehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 7; and (iii) a glyoxylate reductase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0309] 63. The microorganism of any one of paragraphs 52 to 62, which has been genetically engineered to introduce one or more of the following: (i) a gene encoding an alcohol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 6; (ii) a gene encoding an aldehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 8; and (iii) a gene encoding a glyoxylate reductase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 10.

[0310] 64. The microorganism of any one of paragraphs 52 to 63, wherein the microorganism has been genetically engineered to express at least a portion of a pathway for adipic acid utilization, and optionally, one or more genes encoding a long-chain fatty acid CoA ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxybutyryl-CoA dehydrogenase, and / or a 3-oxoadipyl-CoA thiolase.

[0311] 65. The microorganism of any one of paragraphs 52 to 64, wherein the microorganism is genetically engineered to express one or more of: (i) a long-chain fatty acid CoA ligase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 11; (ii) an acyl-CoA dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 13; (iii) an enoyl-CoA hydratase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 15; (iv) a 3-hydroxybutyryl-CoA dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 17; and (v) a 3-oxoadipyl-CoA thiolase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 19.

[0312] 66. The microorganism of any one of paragraphs 52 to 65, which has been genetically engineered to introduce one or more of: (i) a gene encoding a long-chain fatty acid CoA ligase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 12; (ii) a gene encoding an acyl-CoA dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 14; (iii) a gene encoding an enoyl-CoA hydratase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 16; (iv) a gene encoding a 3-hydroxybutyryl-CoA dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 18; and (v) a gene encoding a 3-oxoadipyl-CoA thiolase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 20.

[0313] 67. The microorganism of any one of paragraphs 52 to 66, wherein the microorganism has been genetically engineered to express at least a portion of a pathway for 6-hydroxycaproic acid utilization, and optionally, one or more genes encoding an alcohol dehydrogenase and / or an aldehyde dehydrogenase.

[0314] 68. The microorganism of any one of paragraphs 52 to 67, which has been genetically engineered to express (i) an alcohol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 21; and / or (ii) an aldehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0315] 69. A microorganism described in any one of paragraphs 52 to 68, which has been genetically engineered to introduce: (i) a gene encoding an alcohol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 22; and / or (ii) a gene encoding an aldehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 24.

[0316] 70. The microorganism of any one of paragraphs 52 to 69, wherein the microorganism is genetically engineered to express at least a portion of a pathway for 3-hydroxybutyrate utilization, and optionally, one or more genes encoding acyl-CoA synthetase and / or 3-hydroxybutyrate dehydrogenase.

[0317] 71. The microorganism of any one of paragraphs 52 to 70, which has been genetically engineered to express (i) an acyl-CoA synthetase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 25; and / or (ii) a 3-hydroxybutyrate dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 27.

[0318] 72. The microorganism of any one of paragraphs 52 to 71, which has been genetically engineered to introduce: (i) a gene encoding an acyl-CoA synthetase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 26; and / or (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 28.

[0319] 73. The microorganism of any one of paragraphs 52 to 72, wherein the microorganism is genetically engineered to express at least a portion of a pathway for 3-hydroxyvalerate utilization, and optionally, one or more genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and / or 3-ketoacyl-CoA thiolase.

[0320] 74. The microorganism of any one of paragraphs 52 to 73, which has been genetically engineered to express one or more of: (i) an acyl-CoA synthetase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 25; (ii) a 3-hydroxybutyrate dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 27; and (iii) a 3-ketoacyl-CoA thiolase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0321] 75. The microorganism of any one of paragraphs 52 to 74, which has been genetically engineered to introduce one or more of: (i) a gene encoding an acyl-CoA synthetase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 26; (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 28; and (iii) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 30.

[0322] 76. The microorganism of any one of paragraphs 52 to 75, wherein the microorganism is genetically engineered to express at least a portion of a pathway for 1,4-butanediol utilization, and optionally, one or more genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase.

[0323] 77. (i) a methanol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 31; (ii) a methanol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 33; (iii) an aldehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 23; (iv) an aldehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 35; (v) an alcohol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 37; (vi) a 77. The microorganism of any one of paragraphs 52 to 76, wherein the microorganism is genetically engineered to express one or more of: (i) an alcohol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 5; (ii) an alcohol dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 39; (viii) a succinic semialdehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 41; and (ix) a succinic semialdehyde dehydrogenase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 43.

[0324] 78. (i) a gene encoding a methanol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 32; (ii) a gene encoding a methanol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 34; (iii) a gene encoding an aldehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 24; (iv) a gene encoding an aldehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 36; (v) a gene encoding an alcohol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 38 78. The microorganism of any one of paragraphs 52 to 77, wherein the microorganism has been genetically engineered to introduce one or more of: (i) a gene encoding an alcohol dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 6; (vii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 40; (viii) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 42; and (ix) a gene encoding a succinic semialdehyde dehydrogenase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 44.

[0325] 79. The microorganism of any one of paragraphs 52 to 78, which has been genetically engineered to express at least a portion of a pathway for the synthesis of PHA, and optionally, one or more genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and / or PHA synthase.

[0326] 80. The microorganism of any one of paragraphs 52 to 79, which has been genetically engineered to express one or more of: (i) a 3-ketoacyl-CoA thiolase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 45; (ii) an enoyl-CoA hydratase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 47; and (iii) a PHA synthase having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 49.

[0327] 81. The microorganism of any one of paragraphs 52 to 80, which has been genetically engineered to introduce one or more of the following: (i) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 46; (ii) a gene encoding an enoyl-CoA hydratase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 48; or (iii) a gene encoding a PHA synthase and having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 50.

[0328] 82. The microorganism of any one of paragraphs 52 to 81, which has been genetically engineered by transfection, by transduction, or by gene editing.

[0329] 83. The microorganism of any one of paragraphs 52 to 82, which is a bacterium.

[0330] 84. The microorganism of any one of paragraphs 52 to 83, which is capable of producing PHA utilizing one or more, two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

[0331] 85. A microorganism described in any one of paragraphs 52 to 84, which is capable of producing PHA using each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

[0332] 86. A vector comprising a gene encoding an enzyme having at least 70% sequence identity to any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49.

[0333] 87. A vector comprising a gene comprising or consisting of a nucleotide sequence having at least 70% sequence identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50.

[0334] 88. The vector according to paragraph 86 or 87, which is a plasmid or viral vector.

[0335] 89. A cell comprising the vector described in any one of paragraphs 86 to 88.

[0336] 90. The cell of paragraph 89, which is a microorganism, optionally a bacterium.

[0337] The practice of the present invention will employ, unless otherwise indicated, conventional techniques that are within the capabilities of those skilled in the art. Such techniques are explained in the literature. It should be understood that various changes and modifications to the preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. 1. A method for producing polyhydroxyalkanoates (PHAs) from polyester waste, comprising: (a) providing a culture broth containing polyester waste; (b) culturing a microorganism in the culture broth to produce a PHA, wherein the microorganism utilizes a plurality of polyester monomers from the polyester waste to produce the PHA.

2. 2. The method of claim 1, wherein the microorganism is from the genus Paracoccus, and optionally the microorganism is Paracoccus denitrificans.

3. The microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof, and optionally the microorganism is selected from the group consisting of Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, Paracoccus denitrificans ATCC 19367, Paracoccus denitrificans ATCC 17741, Paracoccus denitrificans ATCC 13543, Paracoccus denitrificans NCIB 8944, Paracoccus denitrificans NRRL B-3785, Paracoccus denitrificans CCM 982, Paracoccus denitrificans LMD 22.21, Paracoccus denitrificans JCM 21484, Paracoccus denitrificans NBRC 102528, and Paracoccus denitrificans NCCB 22021.

4. 4. The method of claim 1, wherein the polyester waste comprises two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers selected from succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxycaproic acid, 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and 1,4-butanediol.

5. The method according to any one of claims 1 to 4, wherein the polyester waste is pre-treated, optionally the polyester waste is mechanically and / or chemically treated.

6. 6. The method according to any one of claims 1 to 5, wherein the microorganism is cultivated under anaerobic conditions, optionally a single microbial strain is cultivated and / or the method comprises a single cultivation step.

7. 7. The method of any one of claims 1 to 6, wherein at least about 50%, at least about 60%, at least about 70%, at least about 75%, or at least about 80% by weight of the polyester waste is utilized during the culturing.

8. 8. The method of any one of claims 1 to 7, wherein the PHA comprises or consists of polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof, optionally wherein the PHA comprises or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

9. A culture broth comprising polyester waste and a microorganism, wherein the microorganism is capable of utilizing a plurality of polyester monomers from the polyester waste to produce a PHA.

10. A polyhydroxyalkanoate (PHA) produced by the method according to any one of claims 1 to 8.

11. An article comprising or consisting of the PHA of claim 10.

12. 1. Use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism produces PHA by utilizing a plurality of polyester monomers from the polyester waste.

13. A microorganism for producing polyhydroxyalkanoates (PHAs) from polyester waste, the microorganism comprising genes encoding pathways for the utilization of multiple polyester monomers and genes encoding pathways for the synthesis of PHAs, and the microorganism is genetically engineered to express at least a portion of one or more of the pathways.

14. A vector comprising a gene encoding an enzyme having at least 70% sequence identity to any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49; and / or a gene comprising or consisting of a nucleotide sequence having at least 70% sequence identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:

50.

15. 15. A cell comprising the vector of claim 14, which is optionally a microorganism, optionally a bacterium.

Images