Biorecycling polyester to PHA

Abstract

The present invention provides a method of producing a polyhydroxyalkanoate (PHA) from polyester waste, the method comprising the steps of: (a) providing a culture solution comprising polyester waste; and (b) culturing a microorganism in the culture solution to produce PHA.

Description

Technical Field

[0001] The present invention relates to a method for producing polyhydroxyalkanoates (PHA) from polyester waste. The present invention also relates to PHA produced by the method and articles prepared using the PHA. Background Art

[0002] Plastics are widely used because of their adaptability, light weight, durability, and flexibility. However, commonly used plastics are not biodegradable. Therefore, they accumulate in landfills or the natural environment without decomposing. As of 2015, approximately 6300 Mt of plastic waste had been generated, of which only about 9% was recycled, 12% was incinerated, and 79% accumulated in landfills or the natural environment (see Geyer, R. et al., 2017, Science advances, Vol. 3, No. 7: p. e1700782).

[0003] The degradation of plastics generally occurs slowly in nature and involves various environmental factors such as temperature, humidity, pressure, and the action of microorganisms. To accelerate degradation, plastic waste can be degraded by physical processes (such as soil burial or combustion) or chemical processes (such as by photo-oxidative hydrolysis or degradation with specific and irritating chemicals). However, both physical and chemical methods have significant drawbacks (see Bano, K. et al., 2017, Current pharmaceutical biotechnology, Vol. 18, No. 5: pp. 429 - 440).

[0004] An alternative to petroleum-based plastics is bio-based polyesters. Polyhydroxyalkanoates (PHA) are structurally diverse microbial polyesters synthesized by many prokaryotic microorganisms. Since PHA are biocompatible, bioabsorbable, and biodegradable, their environmental impact is reduced. When PHA-based products are left in the environment, they are degraded to CO 2 、H 2 O and CH 4 , which is beneficial for the recycling and renewability of the natural cycle. However, compared to petroleum-based plastics, the bacterial synthesis of PHA is currently not cost-effective. Current techniques employ a two-step microbial process that converts organic waste (including PHA waste) to volatile fatty acids (VFA) under anaerobic conditions and then converts the VFA to PHA in a second aerobic fermentation step (see Riaz, S. et al., 2021, Polymers, Vol. 13, No. 2: p. 253).

[0005] Therefore, there is a need for new methods for degrading plastic waste and also for new methods for producing PHA. Summary of the Invention

[0006] The present inventors have developed a direct and efficient method for producing polyhydroxyalkanoates (PHA) from polyester waste.

[0007] The present inventors have demonstrated that the method allows the use of a variety of polyester monomers, including polyester monomers that are difficult to biodegrade, such as 1,4-butanediol. The method allows the use of mixed polyester waste containing biodegradable polyesters (e.g., PHA, PHB, PHBH) and non-biodegradable polyesters (e.g., PET).

[0008] The present inventors have demonstrated that the method can be carried out in a one-step process using a single microorganism for cultivation. This is contrary to the two-step processes currently carried out. The present inventors have also demonstrated that the cultivation can be carried out under aerobic or anoxic conditions. This flexibility can reduce the need for oxygen in the fermenter, which is usually a limiting factor in large-scale fermentation.

[0009] The present inventors have also identified microorganisms with suitable pathways for utilizing mixed polyester waste by constructing a new genomic metabolic model. The present inventors have identified the genes involved in said pathways.

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

[0011] The microorganism can utilize one or more polyester monomers from the polyester waste to produce PHA. The microorganism can utilize a variety of polyester monomers from the polyester waste to produce PHA. Suitably, 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. Suitably, the microorganism utilizes polyester monomers from a variety of polyesters in the polyester waste to produce PHA. Suitably, the microorganism utilizes polyester monomers from at least three, at least four, at least five, at least six, at least seven or at least eight polyesters in the polyester waste to produce PHA. In some embodiments, the microorganism utilizes 1,4-butanediol from the polyester waste to produce PHA.

[0012] Any suitable microorganism can be used in the method of the present invention. Suitably, the microorganism is from the genus Paracoccus. Suitably, the microorganism is Paracoccus denitrificans. Suitably, the microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof. Suitably, 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 NCCB22021, 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 comprise 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 utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol. The microorganism may comprise genes encoding each of: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0014] Any suitable polyester waste can be utilized. Suitably, 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 the following: succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol. Suitably, the polyester waste comprises succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol. In some embodiments, the polyester waste comprises 1,4-butanediol. Suitably, the polyester waste comprises the polyester monomers in free monomer form. 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 selected from the following: polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(succinic acid / terephthalic acid / isophthalic acid butylene glycol ester)-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 can be pretreated. Suitably, the polyester waste is subjected to mechanical treatment and / or chemical treatment.

[0015] The method of the present invention can further include the step of pretreating the polyester waste. Any suitable pretreatment can be used. Suitably, the method further includes the step of mechanically treating the polyester waste (e.g., the polyester waste can be shredded). Suitably, the method further includes the step of chemically treating the polyester waste (e.g., the polyester waste can be subjected to alkali treatment).

[0016] Any suitable culture conditions can be used. Suitably, the culture medium contains an amount of polyester waste from 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. Suitably, the culture medium contains a mineral salt medium. Suitably, the microorganism is cultured under aerobic or anaerobic conditions. In some embodiments, the microorganism is cultured under anaerobic conditions. Suitably, the microorganism is cultured for about one day to about seven days, about two days to about six days, or about three days to about five days. Suitably, a single microorganism strain is cultured. Suitably, the method includes a single culturing step.

[0017] In some embodiments, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 75 wt%, or at least about 80 wt% of 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 polyhydroxybutyrate (PHB) or its copolymers and / or polyhydroxyvalerate (PHV) or its copolymers, or consist of polyhydroxybutyrate (PHB) or its copolymers and / or polyhydroxyvalerate (PHV) or its copolymers. In some embodiments, the PHA comprises polyhydroxybutyrate (PHB) or its copolymers, or consists of polyhydroxybutyrate (PHB) or its copolymers. In some embodiments, the PHA comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0018] The method may further include any other suitable steps. Suitably, the method further includes the step of recovering the PHA.

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

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

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

[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 various polyester monomers from the polyester waste to produce PHA.

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

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

[0025] In another aspect, the present invention provides a cell comprising the vector according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Additional features and advantages of the present invention are described in the following description of presently preferred embodiments, which is given with reference to the accompanying drawings, and these features and advantages will be apparent from the description, wherein:

[0027] Figure 1 is a schematic diagram showing the microbial recycling of polyester-based plastic waste and its related monomers into polyhydroxyalkanoates (PHA) by Paracoccus denitrificans based on the genetic capabilities of Paracoccus denitrificans.

[0028] Figure 2 is a visualization of the metabolic capabilities of Paracoccus denitrificans to convert polyester monomers based on the constructed GSM (genome-scale model). Metabolic pathways for converting different polyester monomers into PHA (PHB) by Paracoccus denitrificans and its related genes and enzymes were identified. Metabolic pathways for utilizing monomers from polymer structural units: A) Central carbon metabolism (TCA cycle) related to PBS (succinic acid), PLA (lactic acid), PHV (3-hydroxyvaleric acid), PBAT (adipic acid), hydroxyhexanoic acid, PHB (3-hydroxybutyric acid), PBS (1,4-butanediol). The arrows also indicate the pathway for producing PHB from central carbon metabolism (TCA cycle) B) Metabolic pathway for assimilating PET (ethylene glycol).

[0029] Figure 3 Shows the biomass formation (dry cell weight of cell population) of Paracoccus denitrificans when different monomers are supplied as the sole carbon source in a 0.3% (weight / volume) medium.

[0030] Figure 4 Shows the amount of poly(hydroxybutyrate-co-valerate) (PHBV) produced by Paracoccus denitrificans when different plastic monomers are used as the sole carbon source.

[0031] Figure 5 Shows A) cell growth of Paracoccus denitrificans and B) consumption of plastic monomers and production of PHB under anoxic conditions.

[0032] Figure 6 shows A) the cell growth of Paracoccus denitrificans and B) the PHB produced from mechanically and chemically pretreated polymers. Detailed Description

[0033] The preferred features and embodiments of the present invention will now be described by way of non - limiting examples. Those skilled in the art will understand that they can combine all the features of the present invention disclosed herein without departing from the scope of the present invention as disclosed.

[0034] It must be noted that, as used herein and in 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" and "consisting of" are synonymous with "including" and "containing", and are inclusive or open - ended and do not exclude additional unrecited members, elements, or steps. The terms "comprising" and "consisting of" also include the term "consisting of".

[0035] Numeric ranges include the numbers defining the range. As used herein, the term "about" means approximately, in the vicinity, roughly, or around. Unless otherwise specified, any nucleic acid sequence is written from left to right in the 5' to 3' orientation; amino acid sequences are written from left to right in the amino - to - carboxyl orientation.

[0036] All publications mentioned in the specification are incorporated herein by reference. The publications discussed herein are provided only for their disclosure prior to the filing date of the present patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims herein.

[0037] a. Method for producing PHA from polymer waste

[0038] In one aspect, the present invention provides a method for producing polyhydroxyalkanoates (PHA) from polyester waste, the method comprising the steps of: (a) providing a culture medium comprising polyester waste; and (b) culturing a microorganism in the culture medium to produce PHA.

[0039] a. Microorganisms for producing PHA from polyester waste

[0040] Any suitable microorganism described herein (e.g., in the section entitled "Microorganisms") can be used to produce PHA from polyester waste. A mixture of microorganisms or a single microorganism can be used. In some embodiments, a single microorganism (e.g., a single microbial strain) is used.

[0041] The microorganism can utilize one or more polyester monomers from the polyester waste to produce PHA. Suitably, the microorganism utilizes 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 to produce PHA. The polyester monomers can be in the form of free polyester monomers, oligopolyesters, or polyesters. Suitably, the polyester monomers are in the form of free polyester monomers or oligopolyesters. Suitably, the polyester monomers are in the form of free polyester monomers.

[0042] In some embodiments, the microorganism utilizes 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 the following from the polyester waste to produce PHA: succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol. In some embodiments, the microorganism utilizes each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol from the polyester waste to produce PHA.

[0043] The microorganism can utilize the polyester monomers of one or more polyesters from the polyester waste to produce PHA. Suitably, the microorganism is capable of utilizing the polyester monomers of 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 to produce PHA.

[0044] In some embodiments, the microorganism utilizes 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 selected from polyester waste to produce PHA: polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), poly(butylene succinate-co-terephthalate) (PBST), poly(butylene succinate / terephthalate / isophthalate)-co-(lactate) (PBSTIL), polybutylene terephthalate (PBT), poly(butylene adipate terephthalate) (PBAT), polyethylene terephthalate (PET), poly(ethylene adipate) (PEA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0045] In some embodiments, the microorganism utilizes 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 the following to produce PHA: polybutylene succinate (PBS), poly(butylene 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 utilizes polyester monomers from one or more, two or more, or three polyesters selected from the following to produce PHA: polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0046] In some embodiments, the microorganism utilizes at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 75 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, at least about 95 wt% of polyester waste.

[0047] b. Polyester waste

[0048] Any suitable polyester waste can be used. As described above, the inventors have demonstrated that the method allows the utilization of a variety of polyester monomers, including polyester monomers that are difficult to biodegrade such as 1,4-butanediol. The method allows the utilization of mixed polyester waste containing biodegradable polyesters (e.g., PHA, PHB, PHBH) and non-biodegradable polyesters (e.g., PET). The polyester waste can be polyester plastic waste.

[0049] A polyester is a polymer that contains an ester functional group in each repeating unit of its main chain. Polyesters can include naturally occurring polymers as well as synthetic polymers. Natural polyesters and some synthetic polyesters are biodegradable, but most synthetic polyesters are non-biodegradable. Polyesters include polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoates (PHA) 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).

[0050] 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; PBAT 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 degraded into their polyester monomers, for example, by hydrolysis cleavage of the ester bond. Hydrolysis cleavage can occur passively or can be catalyzed by a chemical process or an enzymatic process.

[0051] Suitably, the polyester waste contains 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 contains a plurality of polyester monomers. The polyester monomers can be in the form of free polyester monomers, oligopolyesters or polyesters. Suitably, the polyester monomers are in the form of free polyester monomers or oligopolyesters. Suitably, the polyester monomers are in the form of free polyester monomers. The inventors have confirmed that the method of the present invention allows the use of free polyester monomers, oligomers or polymers (e.g., after a pretreatment step).

[0052] In some embodiments, the polyester waste contains 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 the group consisting of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid and 1,4-butanediol. In a preferred embodiment, the polyester waste contains 1,4-butanediol. In some embodiments, the polyester waste contains each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid and 1,4-butanediol (either in the form of free polyester monomers or in the form of polyester polymers).

[0053] Suitably, the polyester waste contains 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 contains a plurality of polyesters.

[0054] In some embodiments, the polyester waste contains 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 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), polyethylene adipate (PEA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) or copolymers thereof.

[0055] 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 the group consisting of: polybutylene succinate (PBS), poly(butylene adipate-co-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 the group consisting of: polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0056] c. Pretreatment

[0057] The polyester waste can be pretreated, i.e., processed, before being added to the culture medium. The pretreated polyester waste can comprise polyester monomers and / or oligomers, especially polyester monomers. The inventors have confirmed that pretreatment can promote the degradation of polyester waste into smaller particles and make the polyester monomers more readily available. Any suitable pretreatment can be used (see, for example, Ragaert, K. et al., 2017, Waste management, vol. 69: pp. 24-58 and WO2002036675A2). Any combination of pretreatment steps can be used, and each step can be repeated one or more times.

[0058] In some embodiments, the method of the present invention comprises the steps of: (a) pretreating the polyester waste; (b) providing a culture medium comprising the pretreated polyester waste; and (c) culturing microorganisms in the culture medium to produce PHA. In some embodiments, the polyester waste is subjected to mechanical treatment and / or chemical treatment (before being added to the culture medium).

[0059] In some embodiments, the polyester waste is subjected to mechanical treatment (before being added to the culture medium). For example, the polyester waste can be separated and / or sorted; baled; washed; ground, shredded, and / or cut; and / or compounded and / or pelletized before being added to the culture medium.

[0060] In some embodiments, the polyester waste is separated and / or sorted (before being added to the culture medium). This can be based on shape, density, size, color, or chemical composition. The polyester waste can be sorted manually or automatically (e.g., by flotation). In some embodiments, the polyester waste is compressed and baled (before being added to the culture medium). If the plastic is not processed when sorted, it is often compressed and baled for transportation purposes. In some embodiments, the polyester waste is washed (before being added to the culture medium). Washing can be used to remove contaminants such as organic contaminants.

[0061] In preferred embodiments, the polyester waste is ground, shredded, and / or cut (before being added to the culture medium). 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 medium). Suitably, the polyester waste can have 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. Suitably, the polyester waste can have 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.

[0062] In some embodiments, the polyester waste is chemically treated (before being added to the culture medium). Chemical treatment can be used to depolymerize the polyester partially or completely into its polyester monomers. Depending on the chemical reagent and the polyester, different depolymerization pathways such as methanolysis, glycolysis, hydrolysis, aminolysis, aminodecomposition, and / or hydrogenation can be used. In some embodiments, the polyester waste is hydrolyzed (before being added to the culture medium). During hydrolysis, the polyester can react with water under neutral or acidic conditions to break the polyester chains. High temperature and / or high pressure can be used to accelerate hydrolysis. In some embodiments, the polyester waste is subjected to alkali treatment (before being added to the culture medium). Suitably, 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), and optionally shaken or stirred at about 200 rpm to about 500 rpm (e.g., about 300 rpm to about 400 rpm). Suitably, the polyester waste is neutralized after alkali treatment. Suitably, the polyester waste is neutralized with an acidic solution (e.g., containing hydrochloric acid). Suitably, the polyester waste is neutralized to about pH 7.

[0063] In some embodiments, the polyester waste is mechanically and chemically treated (before being added to the culture medium). In some embodiments, the polyester waste is ground, shredded, and / or cut and subjected to alkali treatment (before being added to the culture medium).

[0064] d. Culture medium

[0065] In one aspect, the present invention provides a culture medium comprising polyester waste and microorganisms. The microorganisms can be any suitable microorganisms described herein (e.g., in the section entitled "Microorganisms") for producing PHA from polyester waste. The polyester waste can be any polyester waste described herein (e.g., in the sub-section entitled "Polyester Waste"). Suitably, the culture medium may further comprise PHA, suitably any PHA described herein (e.g., in the sub-section entitled "Production of PHA").

[0066] The polyester waste can be added to the culture medium in any suitable amount. Suitably, the culture medium comprises 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 of polyester waste. Suitably, the culture medium comprises 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 of polyester waste. Suitably, the culture medium comprises 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 of polyester waste.

[0067] Any suitable culture medium can be used to culture the microorganisms. Suitably, the pH of the culture medium is about 7 and comprises all the nutrients and trace elements necessary for culturing the microorganisms. The culture medium can depend on the microorganisms used and / or the culture conditions. For example, the optimal culture medium for anaerobic growth of Paracoccus denitrificans is described in Hahnke, S.M. et al., 2014, Frontiers in microbiology, Volume 5: Page 18.

[0068] In some embodiments, the culture medium comprises a mineral salt medium. Suitably, the culture medium comprises at least about 80% (v / v), at least about 85% (v / v), at least about 90% (v / v) or at least about 95% (v / v) of the mineral salt medium. Suitably, the mineral salt medium may comprise about 22.7 g / L of dipotassium hydrogen phosphate, about 0.95 g / L of potassium dihydrogen phosphate, about 0.67 g / L of ammonium sulfate and about 2 mL / L of a trace metal solution. Suitably, the trace metal solution may comprise sodium, zinc, calcium, iron, molybdenum, copper, cobalt, manganese and magnesium.

[0069] e. Culture conditions

[0070] Any suitable culture conditions can be used. As described above, the inventors have confirmed that the method can be carried out by a one-step process using a single microorganism for culturing. The inventors have also confirmed that the culturing can be carried out under aerobic conditions or anaerobic conditions. The culture conditions may depend on the microorganism used.

[0071] Suitably, the microorganism is cultured under aerobic or anaerobic conditions. As used herein, "aerobic" conditions are rich in free oxygen (O 2 ). As used herein, "anaerobic" conditions are characterized by limited free oxygen or lack of free oxygen but contain other electron acceptors (such as ammonium sulfate, potassium nitrate and / or sodium sulfate). Suitably, anaerobic conditions can be produced by replacing oxygen with another gas such as an inert gas (such as helium). Suitably, anaerobic conditions can be produced by helium washing. In some embodiments, the microorganism is cultured under aerobic conditions. In some embodiments, the microorganism is cultured under anaerobic conditions. The microorganism can be cultured partially under aerobic conditions and partially under anaerobic conditions.

[0072] The microorganism can be cultured for any suitable duration. Suitably, the microorganism is cultured for at least about one day, at least about two days, at least about three days, at least about four days or at least about five days. Suitably, the microorganism is 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. Suitably, the microorganism is cultured for about one day to about seven days, about two days to about six days, or about three days to about five days. Suitably, the microorganism is cultured until it reaches 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 of cell biomass.

[0073] The microorganism can be cultured at any suitable temperature. For example, Paracoccus denitrificans can be cultured at a temperature of 11°C - 45°C (Hahnke, S.M. et al., 2014, Frontiers in microbiology, Volume 5: Page 18). Suitably, the microorganism is cultured at about 30°C to about 40°C. Suitably, the microorganism is cultured at about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C or at about 37°C. Suitably, the microorganism is cultured at about 30°C, about 34°C or at about 37°C.

[0074] The microorganism can be cultured under static, shaking or stirring conditions. In some embodiments, the microorganism is cultured under shaking or stirring conditions. In some embodiments, the microorganism is cultured under shaking conditions. Any suitable shaking conditions can be used (see for example W. and Büchs, J., 2012, Trends in biotechnology, Volume 30, Issue 6: Pages 307 - 314). Suitably, the shaking conditions can be about 100 rpm to about 400 rpm, or about 200 rpm to about 300 rpm.

[0075] The culture broth can be inoculated with the microorganism. Any suitable inoculation can be used. Suitably, the inoculum is added in an amount of at least about 1% (v / v) or at least about 2% (v / v). Suitably, the inoculum is added in an amount of about 1% (v / v) or about 2% (v / v). Suitably, the inoculum can be derived from the culture of the microorganism in an enriched medium (such as LB medium).

[0076] The method can include one or more culture steps. In some embodiments, the method includes a single culture step. In some embodiments, the method does not include a second culture step using a second microorganism (or a second mixture of microorganisms). During a single culture step, the culture conditions can be adjusted, for example to lower or raise the temperature, and / or the culture conditions can be monitored and maintained, for example to maintain the pH and / or the minimum level of nutrients. A single culture step can consist of culturing a single microorganism (or a single mixture of microorganisms) in a culture broth. Another culture step can consist of culturing another microorganism (or another mixture of microorganisms) in a culture broth.

[0077] f. Production of PHA

[0078] The type and / or amount of PHA produced can depend on, for example, the polyester waste utilized, the microorganism and / or the culture conditions. Those skilled in the art will be able to optimize the method to obtain the desired type and / or amount of PHA.

[0079] Polyhydroxyalkanoates (PHA) include a group of naturally occurring biodegradable polyesters synthesized by microorganisms and may have the following general formula, where typically x = 1 - 8 and n = 100 to 1000 (see Li, Z. et al., 2016, NPG Asia Materials, Volume 8, Issue 4: pp. e265 - e265):

[0080]

[0081] Over 150 different PHA monomers have been identified, making them the largest group of natural polyesters. Commonly synthesized PHA monomers include: 3 - hydroxybutyrate, 3 - hydroxyvalerate, 3 - hydroxyhexanoate, 3 - hydroxyoctanoate, 3 - hydroxydecanoate, 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 their copolymers with 3 - hydroxyhexanoate (HHx), 3 - hydroxyheptanoate (HH), and / or 3 - hydroxydecanoate (HD).

[0082] The PHA produced by the present invention will vary according to 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 - hydroxyhexanoic acid, and / or 1,4 - butanediol, the resulting PHA may be rich in 3 - hydroxybutyrate monomers. For example, if the polyester waste is rich in 3 - hydroxyvalerate, the resulting PHA may be rich in 3 - hydroxyvalerate monomers.

[0083] The PHA produced by the present invention may comprise a PHA containing 3-hydroxybutyrate monomers, 3-hydroxyvalerate monomers, and / or 3-hydroxyhexanoate monomers, or consist of a PHA containing 3-hydroxybutyrate monomers, 3-hydroxyvalerate monomers, and / or 3-hydroxyhexanoate monomers. The PHA produced by the present invention may comprise a PHA containing 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers, or consist of a PHA containing 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers. The PHA produced by the present invention may comprise a PHA containing 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers, or consist of a PHA containing 3-hydroxybutyrate monomers and / or 3-hydroxyvalerate monomers. The PHA produced by the present invention may contain 3-hydroxybutyrate monomers. In some embodiments, the PHA produced by the present invention contains from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxybutyrate monomers and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxyvalerate monomers, or consists of from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxybutyrate monomers and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxyvalerate monomers. In other embodiments, such as when the polyester waste is rich in 3-hydroxyvalerate, the PHA produced by the present invention contains from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxyvalerate monomers, or consists of from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) 3-hydroxyvalerate monomers.

[0084] The PHA produced by the present invention may comprise poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), 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 poly(3-hydroxybutyrate) (PHB) or its copolymer and / or poly(3-hydroxyvalerate) (PHV) or its copolymer, or consist of poly(3-hydroxybutyrate) (PHB) or its copolymer and / or poly(3-hydroxyvalerate) (PHV) or its copolymer. The PHA produced by the present invention may comprise poly(3-hydroxybutyrate) (PHB) or its copolymer, or consist of poly(3-hydroxybutyrate) (PHB) or its copolymer. The PHB copolymer may comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and / or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH).

[0085] The PHA produced by the present invention may comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or consist of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In some embodiments, the PHBV comprises from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxybutyrate and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxyvalerate, or consists of from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxybutyrate and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxyvalerate. In other embodiments, the PHBV comprises from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxyvalerate monomers, or consists of from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxyvalerate monomers.

[0086] The method of the present invention can be used to produce an amount of PHA 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 method of the present invention can be used to produce an amount of PHA 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.

[0087] g. Recovery, separation and / or purification of PHA

[0088] Once the culturing step is completed, any suitable method known in the art can be used to recover, isolate, and / or purify PHA from the resulting microbial slurry (see, for example, Pagliano, G. et al., 2021, Frontiers in Bioengineering and Biotechnology, Vol. 9: p. 54; Pérez-Rivero, C. et al., 2019, Biochemical Engineering Journal, Vol. 150: p. 107283; and López-Abelairas, M. et al., 2015, Biochemical Engineering Journal, Vol. 93: pp. 250-259).

[0089] In some embodiments, the method of the present invention comprises the steps of: (a) providing a culture medium containing polyester waste; (b) culturing a microorganism in the culture medium to produce a microbial slurry containing PHA; and (c) recovering PHA from the microbial slurry. In some embodiments, the method of the present invention comprises the steps of: (a) pretreating the polyester waste; (b) providing a culture medium containing the pretreated polyester waste; (c) culturing a microorganism in the culture medium to produce a microbial slurry containing PHA; and (d) recovering PHA from the microbial slurry. PHA can be recovered by solvents and / or by cell lysis.

[0090] Suitably, PHA can be recovered from the microbial broth by solvent extraction (see, for example, Pagliano, G. et al., 2021, Frontiers in Bioengineering and Biotechnology, Volume 9: Page 54). Solvent extraction of PHA from the microbial broth can involve the following steps: (i) contacting and mixing the microbial broth with a solvent; (ii) heating the mixture; (iii) separating the extraction residue (non-PHA biomass and water) from the PHA-rich phase (PHA dissolved in the solvent); and (iv) separating PHA from the solvent by evaporative precipitation of PHA.

[0091] Suitably, PHA can be recovered from the microbial broth by cell lysis (see, for example, Pagliano, G. et al., 2021, Frontiers in Bioengineering and Biotechnology, Volume 9: Page 54). Cell lysis can involve the following steps: (i) mixing the microbial broth with an additive (such as an alkali, surfactant, oxidant) to dissolve non-PHA components; (ii) separating solid PHA from the liquid (containing non-PHA components); and (iii) drying and purifying PHA.

[0092] After recovery, PHA can be subjected to one or more additional downstream processing steps such as separation and / or purification (see, for example, Pérez-Rivero, C. et al., 2019, Biochemical Engineering Journal, Volume 150: Page 107283). In some embodiments, PHA is separated. Conventional separation methods include precipitation, crystallization, centrifugation, decantation, filtration, or a combination thereof. In some embodiments, PHA is purified. Suitably, PHA can be purified by washing the crude PHA with a solvent such as ethanol, acetone, ether, or any combination thereof.

[0093] In some embodiments, the method of the present invention comprises the following steps: (a) providing a culture broth containing polyester waste; (b) culturing microorganisms in the culture broth to produce a microbial broth containing PHA; (c) recovering PHA from the microbial broth to provide crude PHA; and (d) separating and / or purifying the crude PHA to provide purified PHA. In some embodiments, the method of the present invention comprises the following steps: (a) pretreating the polyester waste; (b) providing a culture broth containing the pretreated polyester waste; (c) culturing microorganisms in the culture broth to produce a microbial broth containing PHA; (d) recovering PHA from the microbial broth to provide crude PHA; and (e) separating and / or purifying the crude PHA to provide purified PHA.

[0094] b. PHA and products

[0095] In one aspect, the present invention provides a polyhydroxyalkanoate (PHA) obtainable or obtained by a method according to the present invention.

[0096] The PHA may comprise poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or consist of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). PHBV is the most promising biopolymer for replacing petroleum-based plastics, but low process productivity and high selling price constitute the main obstacles to its wide use. In some embodiments, the PHBV comprises from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxybutyrate and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxyvalerate, or consists of from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxybutyrate and from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxyvalerate. In other embodiments, the PHBV comprises from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxyvalerate monomers, or consists of from about 5% to about 10% (such as about 5%, about 6%, about 7%, about 8%, about 9% or about 10%) 3-hydroxybutyrate monomers and from about 90% to about 95% (such as about 90%, about 91%, about 92%, about 93%, about 94% or about 95%) 3-hydroxyvalerate monomers.

[0097] The PHA can be provided in any suitable form, for example, the PHA can be provided in the form of resin, sealant, adhesive, granule, powder, microbead, sphere, tablet, film, pellet, etc.

[0098] In one aspect, the present invention provides an article comprising a polyhydroxyalkanoate (PHA) produced by a method according to the present invention, or consisting of a polyhydroxyalkanoate (PHA) produced by a method according to the present invention.

[0099] PHA can have a wide range of applications. For example, as packaging materials for food and other perishable items (see, e.g., Bugnicourt, E. et al., 2014, eXPRESS Polymer Letters, Vol. 8, No. 11 (2014): pp. 791-808); in the medical and pharmaceutical fields (see, e.g., Valappil, S.P. et al., 2006, Expert Review of Medical Devices, Vol. 3, No. 6: pp. 853-868); and in the transportation industry, such as in automobiles or airplanes. Applications include packaging, molded articles, paper coatings, nonwoven fabrics, adhesives, films, and performance additives.

[0100] Suitably, the PHA produced by the method according to the present invention can be used as packaging materials, plastic bags, tableware, and food containers. In some embodiments, the articles of the present invention are packaging, films, and / or bags. For example, the articles of the present invention can be food packaging, fresh films, covering films, laminated films, wrapping films, heat-shrinkable films, shopping bags, garbage bags, gift bags, and fruit and vegetable bags. In some embodiments, the articles of the present invention are vials, bottles, or containers.

[0101] c. Microorganisms

[0102] In one aspect, the present invention provides a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste. The microorganism can be isolated from its natural environment or produced by a technical process (such as genetic engineering).

[0103] As used herein, "microorganism" or "microbial entity" can refer to microscopic-sized organisms, which can exist in their single-cell form or as cell colonies. Exemplary microorganisms include bacteria, archaea, fungi, and protists.

[0104] Suitably, the microorganism of the present invention may be a bacterium. Suitably, the microorganism of the present invention is from the family Rhodobacteraceae. Suitably, the microorganism of the present invention is from the genus Paracoccus. The species in the genus Paracoccus may include Paracoccus acridae, Paracoccus aeridis, Paracoccus aerius, Paracoccus aestuarii, Paracoccus aestuariivivens, Paracoccus alcaliphilus, Paracoccus alimentarius, Paracoccus alkanivorans, Paracoccus alkenifer, Paracoccus aminophilus, Paracoccus aminovorans, Paracoccus amoyensis, Paracoccus angustae, Paracoccus aquimaris, Paracoccus aurantiacus, Paracoccus baruchii, Paracoccus beibuensis, Paracoccus binzhouensis, Paracoccus bogoriensis, Paracoccus caeni, Paracoccus carotinifaciens, Paracoccus cavernae, Paracoccus chinensis, Paracoccus communis, Paracoccus contaminans, Paracoccus denitrificans, Paracoccus endophyticus, Paracoccus ferrooxidans, Paracoccus fistulariae, Paracoccus fontiphilus, Paracoccus gahaiensis, Paracoccus haematequi, Paracoccus haeundaensis, Paracoccus halophilus, Paracoccus halotolerans, Paracoccus hibisci, Paracoccus hibiscisoli, Paracoccus homiensis, Paracoccus huijuniae, Paracoccus indicus, Paracoccus isoporae, Paracoccus jeotgali, Paracoccus kamogawaensis, Paracoccus kawasakiensis, Paracoccus kocurii, Paracoccus kondratievae, P.P. kondratievae, P. koreensis, P. laeviglucosivorans, P. liaowanqingii, P. lichenicola, P. limosus, P. litorisediminis, P. luteus, P. lutimaris, P. mangrovi, P. marcusii, P. marinus, P. methylutens, P. mutanolytics, P. niistensis, P. nototheniae, P. oceanense, P. onubensis, P. pacificus, P. panacisoli, P. pantotrophus, P. pueri, P. ravus, P. rhizosphaerae, P. salipaludis, P. saliphilus, P. sanguinis, P. sediminilitoris, P. sediminis, P. seriniphilus, P. shandongensis, P. siganidrum, P. simplex, P. solventivorans, P. sordidisoli, P. speluncae, P. sphaerophysae, P. stylophorae, P. subflavus, P. sulfuroxidans, P. suum, P. tegillarcae, P. thiocyanatus, P. thiophilus, P. tibetensis, P. versutus, P. xiamenensis, P. yeei, P. zeaxanthinifaciens, and P.zhejiangensis).

[0105] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans, Paracoccus pantotrophus or Paracoccus versutus. In some embodiments, the microorganism of the present invention is Paracoccus denitrificans or Paracoccus pantotrophus.

[0106] In some embodiments, the microorganism of the present invention is Paracoccus denitrificans. Paracoccus denitrificans is a Gram-negative, spherical, non-motile, denitrifying (nitrate-reducing) bacterium (Kelly, D.P. et al., 2006, International Journal of systematic and evolutionary microbiology, Vol. 56, No. 10: pp. 2495-2500).

[0107] In some embodiments, the microorganism of the present invention 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 DSM415, 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.

[0108] In some embodiments, the microorganism of the present invention 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 or a derivative thereof.

[0109] In some embodiments, the microorganism of the invention is Paracoccus denitrificans DSM 413, Paracoccus denitrificans PD1222, Paracoccus denitrificans CNCM I-5881, or a derivative thereof.

[0110] In a preferred embodiment, the microorganism of the invention is Paracoccus denitrificans DSM 413 or a derivative thereof. Paracoccus denitrificans DSM 413 was deposited at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) before August 22, 1990, and is a type strain. Derivatives of Paracoccus denitrificans DSM 413 may 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 LMD22.21, Paracoccus denitrificans JCM 21484, Paracoccus denitrificans NBRC 102528, and Paracoccus denitrificans NCCB 22021.

[0111] Comparison of 16S rRNA gene sequences is routinely used to ensure the correct placement of Paracoccus strains within the α-3 subgroup of the class Alphaproteobacteria (Kelly, D.P. et al., 2006, International Journal of systematic and evolutionary microbiology, Vol. 56, No. 10: pp. 2495-2500). Suitably, 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, for example, GenBank accession number Y16929.1).

[0112] 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 microbe capable of performing the complete denitrification pathway (Baker, S.C. et al., 1998, Microbiology and Molecular Biology Reviews, Vol. 62, No. 4: pp. 1046-1078). Paracoccus denitrificans PD1222 may also be referred to as Paracoccus denitrificans NCCB97099 (Kelly, D.P. et al., 2006, International Journal of systematic and evolutionary microbiology, Vol. 56, No. 10: pp. 2495-2500).

[0113] In some embodiments, the microorganism of the present invention is derived from DES PRODUITS Paracoccus denitrificans deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15) on September 12, 2022 under the accession number CNCM I-5881 according to the terms of the Budapest Treaty. The deposited strain may be referred to herein as Paracoccus denitrificans CNCM I-5881.

[0114] In some embodiments, the microorganism of the present 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 with Paracoccus denitrificans having GenBank assembly accession number GCA_000203895.1.

[0115] As used herein, a "derivative" of an existing strain may refer to a genetically engineered variant (e.g., one or more genes have been knocked out and / or inserted) or a naturally occurring variant (e.g., where gene drift has occurred). Generally, the derivative may 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.

[0116] a. Utilization of polyester waste

[0117] The microorganisms of the present invention are capable of using one or more polyester monomers to produce PHA. Suitably, the microorganisms are capable of using two or more, three or more, four or more, five or more, six or more, or seven or more polyester monomers to produce PHA.

[0118] In some embodiments, the microorganisms are capable of 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 selected from the group consisting of: succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol to produce PHA. In some embodiments, the microorganisms are capable of using each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol to produce PHA.

[0119] The microorganisms may contain genes encoding one or more pathways for utilizing polyester monomers. Suitably, the microorganisms contain genes encoding two or more, three or more, four or more, five or more, six or more, or seven or more pathways for utilizing polyester monomers.

[0120] In some embodiments, the microorganisms contain genes encoding two or more, three or more, four or more, five or more, six or more, or seven or more pathways selected from the group consisting of: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0121] In some embodiments, the microorganism comprises genes encoding each of the following: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0122] In a preferred embodiment, the microorganism comprises genes encoding a pathway for utilizing 1,4-butanediol. 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 comprises one or more of the following: (i) a gene encoding 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 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; (iii) a gene encoding 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; (iv) a gene encoding 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 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 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 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 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: 41; and (ix) a gene encoding 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 one or more of the following: (i) a gene encoding 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 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 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 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:36; (v) a gene encoding 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 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; (vii) 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:40; (viii) 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: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.

[0123] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing succinic acid. Suitably, the microorganism comprises one or more genes encoding succinate dehydrogenase. In some embodiments, the microorganism comprises: (i) a gene encoding 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 with the amino acid sequence of SEQ ID NO:1. In some embodiments, the microorganism comprises: (i) a gene encoding succinate dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:2.

[0124] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing lactic acid. Suitably, the microorganism comprises one or more genes encoding D-lactate dehydrogenase. In some embodiments, the microorganism comprises: (i) 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 with the amino acid sequence of SEQ ID NO:3. In some embodiments, the microorganism comprises: (i) 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 with the nucleotide sequence of SEQ ID NO:4.

[0125] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing ethylene glycol. Suitably, the microorganism comprises one or more genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, and glyoxylate reductase. In some embodiments, the microorganism comprises one or more of the following: (i) a gene encoding 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 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:7; and (iii) a gene encoding 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 comprises one or more of the following: (i) a gene encoding 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 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 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.

[0126] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing adipic acid. Suitably, 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 comprises one or more of the following: (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; (iii) 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 the amino acid sequence of SEQ ID NO:15; (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 comprises one or more of the following: (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 with 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 with the nucleotide sequence of SEQ ID NO:14; (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%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:16; (iv) a gene encoding a 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 with the nucleotide sequence of SEQ ID NO:18; and (v) a gene encoding a 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 with the nucleotide sequence of SEQ ID NO:20.

[0127] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing 6-hydroxyhexanoic acid. Suitably, 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.

[0128] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing 3-hydroxybutyric acid. Suitably, the microorganism comprises one or more genes encoding acyl-CoA synthetase and 3-hydroxybutyric acid dehydrogenase. In some embodiments, the microorganism comprises: (i) a gene encoding 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 3-hydroxybutyric acid 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 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 3-hydroxybutyric acid 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.

[0129] In some embodiments, the microorganism comprises genes encoding a pathway for utilizing 3-hydroxyvalerate. Suitably, the microorganism comprises one or more genes encoding an acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and 3-ketoacyl-CoA thiolase. In some embodiments, the microorganism comprises one or more of the following: (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 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; and (iii) a gene encoding 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 comprises one or more of the following: (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 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 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.

[0130] Microorganisms may contain genes encoding one or more pathways for the synthesis of PHA. Currently, a total of at least 14 pathways leading to PHA synthesis have been reported. Suitably, the microorganism contains one or more genes encoding PHA synthase. PHA synthase is a key enzyme involved in PHA biosynthesis and acts by polymerizing monomeric hydroxyalkanoate substrates. PHA synthases are classified into four major classes based on their primary sequence, substrate specificity, and subunit composition. Any suitable PHA synthase can be used, such as a native PHA synthase or a genetically engineered PHA synthase (Chek, M.F. et al., 2017, Scientific reports, Vol. 7, No. 1: pp. 1-15).

[0131] Suitably, the microorganism contains one or more genes encoding 3-ketoacyl-CoA thiolase, acetoacetyl-CoA reductase, and PHA synthase. Suitably, the microorganism contains one or more genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and PHA synthase.

[0132] In some embodiments, the microorganism comprises one or more of the following: (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 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:47; and (iii) 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 comprises one or more of the following: (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%, 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: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.

[0133] The microorganism of the present invention is capable of using polyester monomers from one or more polyesters to produce PHA. Suitably, the microorganism is capable of 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 to produce PHA.

[0134] In some embodiments, the microorganism is capable of producing PHA 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: polybutylene succinate (PBS), polybutylene adipate succinate (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), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH). In some embodiments, the microorganism is capable of producing PHA using polyester monomers from each of PBSA, PBST, PBSTIL, PBT, PBAT, PET, PEA, PLA, PCL, PHB, PHBV, and PHBH.

[0135] b. Genetically engineered microorganisms

[0136] In some embodiments, the microorganism has been genetically engineered. The microorganism can be genetically engineered to improve the utilization of one or more polyester monomers and / or improve the synthesis of PHA. This can be achieved, for example, by introducing genes encoding part or all of the pathway, or by optimizing promoters and / or RBSs to increase the expression of part or all of the pathway (see, e.g., Zhang, X. et al., 2020, Trends in biotechnology, vol. 38, no. 7: pp. 689-700). In some embodiments, the microorganism is genetically engineered to overexpress all or part of the pathway.

[0137] The microorganism can be genetically engineered by any suitable method (see, e.g., Keasling, J.D., 1999, Trends in biotechnology, vol. 17, no. 11: pp. 452-460 and Yan, Q. and Fong, S.S., 2017, Frontiers in microbiology, vol. 8: p. 2060). Suitably, the microorganism is genetically engineered by transfection, by transduction, or by gene editing.

[0138] In some embodiments, microorganisms are genetically engineered by transfection. The term "transfection" or "transformation" may refer to a class of genetic engineering in which genes are delivered to target cells using non-viral vectors. Typical bacterial transformation methods may use plasmid DNA.

[0139] In some embodiments, microorganisms are genetically engineered by transduction. The term "transduction" may refer to a class of genetic engineering in which genes are delivered to target cells using viral vectors. Typical bacterial transduction methods may use bacteriophages.

[0140] In some embodiments, microorganisms are genetically engineered by gene editing. The term "gene editing" may refer to a class of genetic engineering in which nucleic acids are inserted, deleted, or replaced in cells. Gene editing can be achieved using engineered nucleases, which can target desired sites in polynucleotides (such as genomes). Such nucleases can generate site-specific double-strand breaks at the desired positions, 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 clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems (see, e.g., Gaj, T. et al. (2013) Trends Biotechnol., Vol. 31: pp. 397-405).

[0141] In some embodiments, the microorganism has been genetically engineered to express one or more of the pathways in the pathway, enhance its expression, and / or overexpress at least a portion thereof. In some embodiments, the microorganism has been genetically engineered to express one or more pathways that utilize polyester monomers, enhance its expression, and / or overexpress at least a portion thereof, and / or the microorganism has been genetically engineered to express one or more pathways for the synthesis of PHA, enhance its expression, and / or overexpress at least a portion thereof.

[0142] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing succinic acid, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress one or more genes encoding succinate dehydrogenase, and / or enhance its expression. In some embodiments, the microorganism has been genetically engineered to express, 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, and / or enhance its expression. In some embodiments, the microorganism has been genetically engineered to introduce a gene encoding 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.

[0143] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing lactic acid, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress one or more genes encoding D-lactate dehydrogenase, and / or enhance its expression. In some embodiments, the microorganism has been genetically engineered to express, 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, and / or enhance its expression. 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.

[0144] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing ethylene glycol, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress one or more genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, and / or glyoxylate reductase, and / or enhance their expression. In some embodiments, the microorganism has been genetically engineered to express, overexpress one or more of the following and / or enhance their expression: (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 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: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 has been genetically engineered to introduce one or more of the following: (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.

[0145] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing adipic acid, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of 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 has been genetically engineered to express, overexpress, and / or enhance the expression of one or more of the following: (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; (iii) 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: 15; (iv) 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 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 has been genetically engineered to introduce one or more of the following: (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; (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%, 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:16; (iv) a gene encoding a 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 a 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.

[0146] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing 6-hydroxyhexanoic acid, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of one or more genes encoding alcohol dehydrogenase and / or aldehyde dehydrogenase. In some embodiments, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of: (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.

[0147] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing 3-hydroxybutyric acid, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of one or more genes encoding acyl-CoA synthetase and / or 3-hydroxybutyric acid dehydrogenase. In some embodiments, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of: (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-hydroxybutyric acid 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 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 3-hydroxybutyric acid 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.

[0148] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing 3-hydroxyvalerate, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of one or more genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and / or 3-ketoacyl-CoA thiolase. In some embodiments, the microorganism has been genetically engineered to express, overexpress, and / or enhance the expression of one or more of the following: (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) 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; 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 has been genetically engineered to introduce one or more of the following: (i) a gene encoding 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 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 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.

[0149] In some embodiments, the microorganism has been genetically engineered to express a pathway for utilizing 1,4-butanediol, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress one or more genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase, and / or enhance their expression.In some embodiments, microorganisms have been genetically engineered to express, overexpress, and / or enhance the expression of one or more of the following: (i) 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) 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; (iii) 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; (iv) 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) 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) 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) 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) 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:41; and (ix) 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 has been genetically engineered to introduce one or more of the following: (i) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:32; (ii) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:34; (iii) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:24; (iv) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:36; (v) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:38; (vi) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:6; (vii) 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 with the nucleotide sequence of SEQ ID NO:40; (viii) 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 with 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 with the nucleotide sequence of SEQ ID NO:44.

[0150] In some embodiments, the microorganism has been genetically engineered to express a pathway for synthesizing PHA, enhance its expression, and / or overexpress at least a portion thereof. Suitably, the microorganism has been genetically engineered to express, overexpress one or more genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and / or PHA synthase and / or enhance their expression. In some embodiments, the microorganism has been genetically engineered to express, overexpress one or more of the following and / or enhance their expression: (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 with the amino acid sequence of SEQ ID NO:45; (ii) 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 with the amino acid sequence of SEQ ID NO:47; and (iii) 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 with the amino acid sequence of SEQ ID NO:49. In some embodiments, the microorganism has been genetically engineered to introduce one or more of the following: (i) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:46; (ii) a gene encoding enoyl-CoA hydratase 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 with the nucleotide sequence of SEQ ID NO:48; and (iii) a gene encoding 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 with the nucleotide sequence of SEQ ID NO:50.

[0151] d. Vectors

[0152] In one aspect, the present invention provides a vector that contains a gene encoding an enzyme for producing polyhydroxyalkanoate (PHA) from polyester waste.

[0153] "Vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention and by way of example, some vectors for recombinant nucleic acid technology allow entities such as nucleic acid segments to be transferred into target cells. Vectors can be used to maintain heterologous nucleic acids within a cell, thereby facilitating the replication of a vector containing a nucleic acid segment, or the expression of a protein encoded by the nucleic acid segment.

[0154] The vector can be a non-viral vector or a viral vector. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, cosmids, chromosomes, artificial chromosomes, and viruses. The vector can be single-stranded or double-stranded. The vector can be naked nucleic acid (e.g., DNA). The vectors used in the present invention can be, for example, naked nucleic acid, plasmids, or viral vectors.

[0155] In one embodiment, the vector is a plasmid. "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 small circular double-stranded DNA molecules in bacteria; however, plasmids are sometimes also present in archaea and eukaryotic organisms.

[0156] In one embodiment, the vector is a viral vector. Viral vectors were initially developed as an alternative to transfecting naked DNA for molecular genetics experiments. Compared to traditional transfection methods, transduction efficiency may be higher and some viruses integrate into the cell genome, thereby facilitating stable expression. Suitably, the viral vector is a bacteriophage.

[0157] a. Succinate pathway

[0158] In one embodiment, the vector contains one or more genes encoding all or part of the pathway for utilizing succinic acid. Suitably, the vector contains a gene encoding succinate dehydrogenase.

[0159] In one embodiment, the vector contains 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 contains a gene encoding succinate 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 with SEQ ID NO:1. In one embodiment, the vector contains such a gene that contains 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 with SEQ ID NO:2, or consists 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 with SEQ ID NO:2.

[0160] MPKPDLPDLPDTCDLLVVGSGAAGMAAAISAHHHGLKPVIVEKSEFFGGSTAVSGGAIWVPCNPIAAAAGMTDDREAARAYIRGETGNRFNAELVDAFLDKSPEAIGFFHERTALKMAHRALSPDYHSDAPGATEGGRALDALDYDGRRLGADLYRMRPPIADFTILGGMPLGRPDIFHFLRMTRSVKSAAYATGAVLRYFRDRLTWGRNTRLVMGAAVSGRLAETVFARNIPLFTGHELVRLLQDESGRVVGAELKGPRGVCRIAAHRGVVLAAGGYPHDAARRAQSFEHVRRGLPHYSMSPVSGTGGGIAAAEAVGAAFVDTNPNAGFWTPVSLLRNADGSVRPFPHLFLDRAKPGVIAVGHDGRRFVNEASSYHDFVQGLIAKLLADGEKSAWLVADHRAMRRYGLGAAHAFPARIGRHVASGYLKRDATLEGLARQCGIDVATFRQTVALFNEAAARGEDPAFGKGSTSYQRYLGDGENRPNPCLRPLEGPFYAVEIYPGDIGTSMGLDITAKGEVRDSRGRTIPGLYACGNDINSVMSGAYPGPGITLGPALTFGYVIGQSAAA

[0161] Exemplary succinate dehydrogenase (SEQ ID NO:1)

[0162]

[0163] Exemplary succinate dehydrogenase gene - peg.3652 (SEQ ID NO:2)

[0164] b. Lactate pathway

[0165] In one embodiment, the vector comprises one or more genes encoding all or part of a lactate utilization pathway. Suitably, the vector comprises a gene encoding D-lactate dehydrogenase.

[0166] 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 with SEQ ID NO:3. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:4, or consists 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 with SEQ ID NO:4.

[0167] MTRPGQAEAAADRTALLDRLRRIVGPAHVLTADRATRRYTRGFRYGEGPVAAVVRPGSLVQMWRVLNAAVASGRAVILQAANTGLTGGSTPWGQDYDREIVLVSVMRLRGIHLIGAGEQVLCLPGATLDALEKRLRPLGREPHSVIGSSCIGASVLGGICNNSGGALIQRGPAYTEMSLYAEVREDGSVALVNHLGLDLGDDPEEILARVEAGELPAPAPTDAWASDREYADHVRDIEAETPARFNADPRRLHESSGCAGKLAVFAVRLDTFQAEKDTAVFYVGSNDPDELTEIRRHILAHFQSLPIAGEYIHREAYDIAAKYGKDTFLFIRHAGTDRMPAFFAAKARMDALTERLGLGATLSDRLAQGVAALMPQHLPRRMNDFRDRYEHHLLLRMGGAGIAEARDYLGAIFPSASGAMFECTPDEGKAAFLHRFAVAGAAVRYRAIHAREVQDIVALDIALRRNDRDWVERLPPDLDAKLEKKLYYGHFFCHVFHQDYVVKKGQDCLAVEHEMWRLLDRRGAEYPAEHNVGHLYHAKPELAGFYRQLDPTNSLNPGIGQTSKCAHWH

[0168] Exemplary D-lactate dehydrogenase (SEQ ID NO:3)

[0169]

[0170] c. Ethylene glycol pathway

[0171] In one embodiment, the vector comprises one or more genes encoding all or part of the glycol utilization pathway. Suitably, the vector comprises genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, and glyoxylate reductase.

[0172] In one embodiment, the vector comprises a gene encoding alcohol dehydrogenase. Alcohol dehydrogenase (EC 1.1.1.1) is an enzyme that can catalyze the conversion of an alcohol to an aldehyde or a ketone (e.g., convert ethylene glycol to glycolaldehyde). In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:5. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:6, or consists 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 with SEQ ID NO:6.

[0173] MTFRANWSYPTTIKFGPGRVTELAEHCRAVGIARPLLVTDKALASLPITAQALDVLDASGLGRAVFSEVDPNPHEGNMEAGIAAYKAGGHDGVICFGGGSALDLGKMIALMADQTVSVWDLEDIGDWWTRADAGKIAPIIAVPTTAGTGSEVGRAGVLTNSATHKKKIIFHPRLMPAVTICDPELTVGMPKFITAGTGMDAFAHCLEAFCSPHYHPMSQGIALEGLRLVNEYLPRAYATPDDLEARAHMMSAAAMGAVAFQKGLGAIHSLSHPVGAVYGTHHGTTNAVVMPMVLDFNRSAIEDRLARAADYLGIKGGFDGFRARVIQLRSELAIPQNLTRLGVQTERLDELTEMALEDPSCGGNPVEMTRENTRALFESCM

[0174] Exemplary alcohol dehydrogenase (SEQ ID NO:5)

[0175]

[0176] Exemplary alcohol dehydrogenase gene - peg.4723 (SEQ ID NO:6)

[0177] In one embodiment, the vector comprises a gene encoding 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 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 with SEQ ID NO:7. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:8, or consists 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 with SEQ ID NO:8.

[0178] MSKTIALISPATGRTLVERQTLGIEDARAAVARARAAQPEWAALPLDERIARIRAGIEALNAMKDAIVPELADQMGRPIRYGGEFGGVNERAGHMMKIAAQALAPTVVEDSDHFAREIRREPVGVVFVIAPWNYPFLTAVNTVVPALVAGNAVILKHASQTLLAGERLAEALHRGGVPAEVMQNVVLDHQTTEALIAGRSFGFVNFTGSVAGGRAIERAAAGTFTATGLELGGKDPGYVRADADLDAAVDGLMDGAMFNSGQCCCGIERIYVHESLFDAFVAKAVDWVNAQKLGNPRDPDTTMGPMAHRRFADLVRAQVSEAVAQGARPLIDPANFPADDGGAYLAPQVLVDVTHDMRVMREESFGPVVGIMPVRDDAEAIGLMNDCDYGLTASIWTADADAAARIGSRLETGTVYMNRCDYLDPALCWTGCKDTGRGAALSGLGYLAVTRPKSYHLKKVTK

[0179] Exemplary aldehyde dehydrogenase (SEQ ID NO:7)

[0180]

[0181] Exemplary aldehyde dehydrogenase gene - peg.4722 (SEQ ID NO:8)

[0182] In one embodiment, the vector comprises a gene encoding glyoxylate reductase. Glyoxylate reductase (EC1.1.1.79) is an enzyme that can catalyze the conversion of glycolate to glyoxylate. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:9. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:10, or consists 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 with SEQ ID NO:10.

[0183] MPAVHATDPTRSRLKVTVTRRLPEAVETRMSELFDVSLNAEDRRMSREELVAAMRVSDVLVPTITDHIDAAMLAQAGDRLKLIANYGAGVDHVDVHSARQRGILVSNTPGVVTEDTADVVMALILGVTRRLPEGMAEMQAGRWQGWSPTAHLGGRLGGRRLGILGMGRIGQAVARRANAFGMQVHYHNRRRLRPEVEAELQATYWESLDQMLARMDIVSVNAPHTPSTFHLLNARRLKLLKPSAVVINTSRGEVIDENALTRMLRAGEIAGAGLDVFEHGHEINPRLRELPNVVLLPHMGSATIEGRVEMGEKVIINIKTFADGHRPPDLVVPSML

[0184] Exemplary glyoxylate reductase (SEQ ID NO:9)

[0185]

[0186] Exemplary glyoxylate reductase gene—peg.1987 (SEQ ID NO:10)

[0187] d. Adipic acid pathway

[0188] In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for utilizing adipic acid. Suitably, the vector comprises 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.

[0189] In one embodiment, the vector comprises a gene encoding a long-chain fatty acid-CoA ligase. A 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 with SEQ ID NO:11. In one embodiment, the vector comprises a gene comprising 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 with SEQ ID NO:12, 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 with SEQ ID NO:12.

[0190] MTPKGRTGGKGMARFASVADRDAVEAEMPYAERQVPHTVYQALTETRDRHPQRPAISFQLFSDPKAPARTLTWTELHERVTETANLFRSLGVGPDDVVAYLLPNCIEAPVVLLAGATAGIVNPINPLLEPDHIAAILRETGAKVLVTLKSFPKSEVAQKAADAVAQAPNVQTVLEVDLRGYLTGVKRLLVPLMRPKVTARHHAKVMDFDAAASAQKHNRLTFDEPAEDRVAAFFHTGGTTGMPKVAQHKQSGMIYNGWLGGTLLFTETDVLMCPLPMFHVFAAYPVLMSCLMSGAQLVMPTPAGYRGEGVFDNFWKLIERWQATFLITVPTAIAALMQRPVNADVSSLKTAISGSAPLPIELYNRFKAATGVEIAEGYGLTEATCLVSCNPINGLKKVGSVGIPLPHTHVRILQRRNGGFHECATDEIGEICVANPGVFEGSTYTEADKNHDLFAESRFLRTGDLGRMDADGYLWITGRAKDLIIRGGHNIDPAEIEDALLSHPKVAAVAAIGQPDSFAGELPCAYVELIAGAEVGLDELMEHARTHIHERAAVPKHVEILPELPKTTVGKIFKPDLRKLAIRRVYDSALAEAGLAAEVGEVVDDRKRGLVAHIRPKGQVDRSAVEQLLGQYALPWEWVG

[0191] Exemplary long-chain fatty acid-CoA ligase (SEQ ID NO:11)

[0192]

[0193] Exemplary long-chain fatty acid-CoA ligase gene—peg.2056 (SEQ ID NO:12)

[0194] 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., convert 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 with SEQ ID NO:13. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:14, or consists 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 with SEQ ID NO:14.

[0195] MSLDPETLAQFLETLDRFVRERLIPNEERVADGDAIPPELVQEIREMGLFGMSIPEEHGGIGLTMAEEVQAALVLGQASPVFRSLVGTNNGIGSQGIIIDGTPEQKAHYLPQLASGEMIASFALTEPDAGSDAGSLRCSARLDGDHYVLNGTKRFITNAPHAGLFTVFARTDPDSKSAAGVTAFLVEAGTPGLHLGPRDRKMGQKGSHTCDVILEDCRVPASAIIGGPDRLGQGFKTAMKVLDRGRLHISAVCVGAAERLIRDSLAYAMERRQFGEPIAEKQLVQAMLADSRAEAYAARCMIEETARRKDAGLSVSTEAACCKMYASEMVGRVADRAVQIHGGAGYMAEYAVERFYRDVRLFRIYEGTTQIQQLVIARNMIREASG

[0196] Exemplary acyl-CoA dehydrogenase (SEQ ID NO:13)

[0197]

[0198] Exemplary acyl-CoA dehydrogenase gene—p.200 (SEQ ID NO:14)

[0199] In one embodiment, the vector comprises a gene encoding 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 of 2-trans / cis-enoyl-CoA. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:15. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:16, or consists 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 with SEQ ID NO:16.

[0200] MRDISQLNLTHLLFDMDGDGIATVTLNRAAKRNALNAETIEELVAVFSALPASGARAVVLRAEGPHFCAGLDLVEHGREERSPAEFMRICLRWHEAFNKIEYGGIPVIAALKGAVVGGGLELASSVHIRVMDETTYFGLPEGQRGLFTGGGATIRVPRLIGQARMMDMMLTGRLYSGDEAVQVGLAQYRVADSEAQAYDLARRVAQNTPLSNFAVCSAISHMQNMSGLDAAYAEAMVAGIVNTQDAARGRLDSFAQGTAQKIKPGEAG

[0201] Exemplary enoyl-CoA hydratase (SEQ ID NO:15)

[0202] atgcgcgacatttcgcagttgaacctgacccacctgctcttcgacatggacggggacggcatcgccaccgtcaccctgaaccgcgccgccaagcgcaacgccctgaatgccgagacgatcgaggaactggtcgcggtcttttccgccctgcccgcctcgggcgcccgtgccgtggtgctgcgcgccgaggggccgcatttctgcgccgggctggacctggtcgagcacgggcgcgaggaacgcagccctgccgagttcatgcgcatctgcctgcgctggcacgaggcgttcaacaagatcgaatatggcggcattccggtcatcgccgcgctcaagggcgcggtggtgggcggcgggctggaactggcctcgtcggtccatatccgggtgatggacgagaccacctatttcggcctgcccgaggggcagcgcgggctgttcaccggcggcggcgccacgatccgcgtgccccggctgatcggccaggcgcgcatgatggacatgatgctgaccggccggctgtattccggggacgaggcggtgcaggtcgggctggcgcaataccgcgtggccgacagcgaggcgcaggcctacgaccttgcccgccgggtggcgcagaacacacccctgtcgaatttcgcggtctgctcggcgatctcgcatatgcagaacatgtcggggctggacgccgcctatgccgaggccatggtcgccggcatcgtcaacacccaggacgccgccaggggaaggctggacagctttgcccagggcacggcgcaaaagatcaagccgggcgaggcaggctga

[0203] Exemplary enoyl-CoA hydratase gene - peg.2628 (SEQ ID NO:16)

[0204] In one embodiment, the vector contains 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 contains 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 with SEQ ID NO:17. In one embodiment, the vector contains such a gene that contains 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 with SEQ ID NO:18, or consists 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 with SEQ ID NO:18.

[0205] MAIQSVGVIGAGQMGNGIAHVFALAGYDVLMTDISREALDKAVAQIDHNLERQVSRGKVSAEDKAAAMRRITTTMTLSDLGKTDLIIEAATERETVKQAIFEDLLPHLKPETILTSNTSSISITRLASRTDRPERFMGFHFMNPVPVMQLVELIRGIATNEETYKALVEVVEKIGKTSASAEDFPAFIVNRILMPMINEAVYTLYEGVGSVKSIDQSMKLGANHPMGPLELADFIGLDTCLAIMNVLHEGLADTKYRPCPLLVKYVEAGWLGRKTGRGFYDYSGEEPVPTR

[0206] Exemplary 3-hydroxybutyryl-CoA dehydrogenase (SEQ ID NO:17)

[0207] atggcgattcaatcggtgggcgtgatcggcgccggacagatgggcaatggcatcgcgcatgtctttgccctggcgggttatgacgtgctcatgaccgacatctcgcgcgaggcgctggacaaggccgtggcgcagatcgaccacaacctggaacgccaggtcagccgcggcaaggtctcggccgaggacaaggccgcggcgatgcggcgcatcaccaccaccatgacgctttccgacctgggcaagaccgacctgatcatcgaggccgccaccgagcgcgagaccgtcaagcaggcgatcttcgaggatctgctgccgcatctgaagcccgagaccatcctgacctcgaacacctcgtcgatctcgatcacccgccttgccagccgcaccgaccggcccgagcgcttcatgggcttccacttcatgaacccggttccggtcatgcagctggtcgagctgatccgcggcatcgccaccaacgaggagacctacaaagccctggtcgaagtggtcgaaaagatcggcaagacctcggccagcgccgaggatttcccggccttcatcgtcaaccgcatcctgatgccgatgatcaacgaggcggtctatacgctttacgagggcgtcggctcggtcaagtccatcgaccagtcgatgaagctgggcgccaaccacccgatggggccgctggaactggcggatttcatcggcctcgacacctgcctggcgatcatgaacgtgctgcacgaggggctggcggacacgaaataccggccctgcccgctcttggtgaaatatgtcgaggcaggctggctgggccgcaagaccgggcgtgggttctacgactattcgggcgaggagccggtgccgacgcgatag

[0208] Exemplary 3-hydroxybutyryl-CoA dehydrogenase gene - peg.2620 (SEQ ID NO:18)

[0209] 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 with SEQ ID NO:19. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:20, or consists 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 with SEQ ID NO:20.

[0210] MTEVFICDYIRTPIGRFGGALSSVRADDLGAIPLRALMARHAGLDWQAVDDVIYGCANQAGEDNRNVARMSALLAGLPVEVPGTTINRLCGSGMDAVLVAARQIAAGEAELMIAGGVESMSRAPFVLPKAESAFSRHAEIHDTTIGWRFVNPAMHAAYGTDSMPQTGQNVADDYGISREAQDAMALASQQKAAAAIASGRLAAEIAPITIPQRKGEPIVVDTDEHPRATTPEALAKLRPLFPNGSVTAGNASGVNDGAAALILASEAAARKHGLTPIARVLGGATAGVPPRIMGIGPAPASQKLMDRLGLTPADFDVIELNEAFAAQGLATLRQLGIADDDPRVNPNGGAIALGHPLGMSGARITGTAALELALTGGKRSLSTMCIGVGQGIAVALERV

[0211] Exemplary 3 - oxoadipyl - CoA thiolase (SEQ ID NO:19)

[0212]

[0213] Exemplary 3-oxoadipyl-CoA thiolase gene—peg.5206 (SEQ ID NO:20)

[0214] e. 6-Hydroxyhexanoic acid pathway

[0215] In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for utilizing 6-hydroxyhexanoic acid. Suitably, the vector comprises a gene encoding an alcohol dehydrogenase and / or an aldehyde dehydrogenase.

[0216] In one embodiment, the vector comprises a gene encoding an alcohol dehydrogenase. The alcohol dehydrogenase can convert 6-hydroxyhexanoic acid to 6-oxohexanoic 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 with SEQ ID NO:21. In one embodiment, the vector comprises a gene comprising 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 with SEQ ID NO:22, 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 with SEQ ID NO:22.

[0217] MAKTMKAAVVREFGKPLTIDEVPVPEPGPGMIQVRIQASGVCHTDLHAAEGDWPVKPNPPFIPGHEGVGFVSAVGAGVKHVKEGDRVGVPWLYTACGHCRHCLGGWETLCESQLNTGYSVNGGFADYVVADPNYVGHLPKNVDFLDIAPVLCAGVTVYKGLKVTDTKPGDWVVISGIGGLGHMAVQYAKAMGMNVAAVDIDDEKLALARKLGATVTVNAATEPDPAAAIRKQTDGGAQGVLVTAVGRKAFEQAIGMVARGGTVALNGLPPGDFPLDIFGMVLNGITVRGSIVGTRLDLQESLDFAGDGKVKATVHKAKLEDINNIFGQMHKGQIEGRMVLDMAG

[0218] Exemplary alcohol dehydrogenase (SEQ ID NO:21)

[0219]

[0220] Exemplary alcohol dehydrogenase gene - peg.2426 (SEQ ID NO:22)

[0221] In one embodiment, the vector comprises a gene encoding an aldehyde dehydrogenase. The aldehyde dehydrogenase can convert 6 - oxohexanoic 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 with SEQ ID NO:23. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:24, or consists 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 with SEQ ID NO:24.

[0222] MPNDQTHPFRGVNALPFEERYDNFIGGEWVAPVSGRYFTNTTPITGAEIGQIARSEAGDIELALDAAHAAKEKWGATSPAERANIMLKIADRMERNLELLATAETWDNGKPIRETMAADLPLAIDHFRYFAGVLRAQEGSISQIDDDTVAYHFHEPLGVVGQIIPWNFPLLMACWKLAPAIAAGNCVVLKPAEQTPAGIMVWANLIGDLLPPGVLNIVNGFGLEAGKPLASSNRIAKIAFTGETTTGRLIMQYASENLIPVTLELGGKSPNIFFADVAREDDDFFDKALEGFTMFALNQGEVCTCPSRVLIQESIYDKFMERAVQRVQAIKQGDPRESDTMIGAQASSEQKEKILSYLDIGKKEGAEVLTGGKAADLGGELSGGYYIEPTIFRGNNKMRIFQEEIFGPVVSVTTFKDQAEALEIANDTLYGLGAGVWSRDANTCYRMGRGIKAGRVWTNCYHAYPAHAAFGGYKQSGIGRETHKMMLDHYQQTKNMLVSYSPKKLGFF

[0223] Exemplary aldehyde dehydrogenase (SEQ ID NO: 23)

[0224]

[0225] Exemplary aldehyde dehydrogenase gene—peg.2425 (SEQ ID NO:24)

[0226] f. 3-Hydroxybutyric acid pathway

[0227] In one embodiment, the vector comprises one or more genes encoding all or part of a pathway for utilizing 3-hydroxybutyric acid. Suitably, the vector comprises a gene encoding an acyl-CoA synthetase and / or a 3-hydroxybutyric acid dehydrogenase.

[0228] 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., convert 3-hydroxybutyric acid to 3-hydroxybutyryl-CoA and / or convert 3-hydroxypentanoic acid to 3-hydroxypentanoyl-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 with SEQ ID NO:25. In one embodiment, the vector comprises a gene comprising 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 with SEQ ID NO:26, 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 with SEQ ID NO:26.

[0229] MSATTLSRSRPGYEQAVAQFRIEDAIAGLRGDLETGLNACVECCDRHCGADRVALRCLSADEALVEYTFEDLRALSARAANLMRDKGIRPGDVVAGLLPRTVELVATVLGAWRLGAVYQPLFTAFGPKAIEHRLKTSGAKLVVTNAAQRPKLDEVEDCPLVATLRGDGPLAPGDVDFREALAMASDQFEPVMRRGEDLFMMMSTSGTTGLPKGVPVPLRALLAFGAYMRDAIDLRETDVFWNIADPGWAYGLYYALTGPLLLGQPTILYEGGFTAETTYRIIERMGVTSLAGSPTAYRLLIAAGPEAAGAVKGRLRVVSSAGEPLNPEIIRWFGEHLAVPIHDHYGQTEMGMCVNNHHGLEHPVRPGSAGLAMPGYRIVVLDDDGNELGPNQPGVLAIDMKRSPLMWFSGYLNQATPALAGGYYRTGDSVEFEPDGSISFIGRSDDVITSSGYRIGPFDVESALIEHPAVVEAAVVGVPDPERTEIVKAFVVLAKGVEGTEALREELAQHVKKRLSAHAYPRMIDFVADLPKTPSGKIQRFVLRKAEVEKLARE

[0230] Exemplary acyl-CoA synthetase (SEQ ID NO:25)

[0231]

[0232] Exemplary acyl-CoA synthetase gene — peg.2994 (SEQ ID NO:26)

[0233] 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-oxovalerate. 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 with SEQ ID NO:27. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:28, or consists 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 with SEQ ID NO:28.

[0234] MFEKFLSGKTAVVTGSNSGIGLGIAHELARAGADLVLNSFTDMPEDHALAESLAAEHGVEVRYVQADMSKGADCRALIEKAGACDILVNNAGIQHVAPIPDFPGEKWDAIIAINLSSAFHTTAAALPLMRKAGWGRVINIASAHGLTASEYKSAYVAAKHGIVGLTKVTALETAKEPITCNAICPGYVLTPIVEKQIPDQMKTHNMSREDVIAKVMLQRQPSGQFATVEQMGGTAVFLCSPAAEQITGTTISVDGGWTAL

[0235] Exemplary 3-hydroxybutyrate dehydrogenase (SEQ ID NO:27)

[0236] atgttcgagaaattcctcagcggcaagacggcggtggtgacgggctccaattcggggatcgggctcgggatcgcgcatgaactggcgcgcgcgggcgctgatctcgtgctgaacagctttaccgacatgcccgaggaccacgcccttgccgaaagccttgccgccgagcatggcgtcgaggtgcgctatgtccaggccgacatgtccaagggcgccgactgccgcgccctgatcgaaaaggccggcgcctgcgacatcctggtgaacaatgccggcatccagcatgtcgcaccgatcccggatttcccgggcgagaaatgggatgcgatcatcgccatcaacctgagttccgcctttcacaccacggcggcggcgctgcccctgatgcgcaaggcaggctgggggcgggtgatcaacatcgcctcggcgcacgggctgacggccagcgaatacaaatcggcctatgtcgcggccaagcacggcatcgtcggcctgaccaaggtgacggcgctggagaccgcgaaggagccgatcacctgcaacgccatctgccccggctatgtgctgaccccgatcgtggaaaagcagatccccgaccagatgaagacccacaacatgagccgcgaggacgtgatcgccaaggtcatgctgcagcgccagccctcggggcaattcgccacggtcgagcagatgggcggcacggcggtcttcctgtgctcgccggcggcggagcagatcaccggcacgaccatctcggtggacggggggtggacggcgctttag

[0237] Exemplary 3-hydroxybutyrate dehydrogenase gene - peg.960 (SEQ ID NO:28)

[0238] g. 3-Hydroxyvaleric acid pathway

[0239] In one embodiment, the vector comprises one or more genes encoding all or part of a pathway utilizing 3-hydroxyvalerate. Suitably, the vector comprises genes encoding acyl-CoA synthetase, 3-hydroxybutyrate dehydrogenase, and / or 3-ketoacyl-CoA thiolase.

[0240] 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 cleave 3-oxovaleryl-CoA into acetyl-CoA and propionyl-CoA. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO: 29. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO: 30, or consists 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 with SEQ ID NO: 30.

[0241] MSENEIVILSGARTAIGTFGGSLAGVPPIQLAATVTRAAIERAGIGPERIGTVVFGHVLNTEPRDMYLSRVAMLDAGVPDTTPAMNVNRLCGSGAQAIVSATQALILGDADFAVAGGAESMSRAPYAVPAARFGAKMGDVQMLDMMVGALTCPMGTGHMGVTAENVAREHDISRQAQDEFALESQKRAAAAIAEGRFKEQIVPIEIKTRKGMVAFDTDEHPKATDLEKLAGLKAVFQKDGTVTAGNASGINDGAAALVLARADAARAAGAKPLFRVLGYAVAGVRPEVMGIGPVPAVEALLKSTGLKIGEFDVIESNEAFAAQALAVNKGLGLDPAKVNPNGGAIALGHPVGATGALVTVKAMYELMRTGGSKGLITMCIGGGQGIALAIERI

[0242] Exemplary 3-ketoacyl-CoA thiolase (SEQ ID NO:29)

[0243]

[0244] h. 1,4-Butanediol pathway

[0245] In one embodiment, the vector comprises one or more genes encoding all or part of the pathway for utilizing 1,4-butanediol. Suitably, the vector comprises genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase.

[0246] 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 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 with SEQ ID NO:31. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:32, or consists 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 with SEQ ID NO:32.

[0247] MKNLMNGACLALLMSGTAALANDSVLAEIAKPQQWAIQMGDYANTRYSTLDQINKDNVKDLRVAWTFSTGVLRGHEGSPLVIGDVMYVHTPFPNRVFALDLNDNGKILWRYEPQQDPNVIAVMCCDTVYRGLSYADGMILLGQADTTVVALDATSGEVKWSTKIGDPGIGETLTATVVPVKDKVLVGISGGEYGVRGRMTALNLTDGSEAWKAWSTGPDEELLVDPETTTHLGKPIGADSSLNSWEGDQWQIGGGTIWGWFSYDPDLNLVYYGTGNPSTWNPSQRPGDNKWSMTIMARDADTGMAKWFYQMTPHDEWDYDGVNEMILTNQTVDGQERKLLTHFDRNGLAYTMDRETGELLVAEKYDPVVNWTTGVDMDPNSETYGRPAVVAEYSTAQNGEDENTTGVCPAALGTKDQQPAAFSPKTNLFYVPTNHVCMDYEPFRVAYTAGQPYVGATLSMYPAPNSHGGMGNFIAWDNTTGEIKWSVPEQFSVWSGALATAGDVVFYGTLEGYLKAVDAQTGEELYKFKTPSGIIGNVMTYEHGGKQYVGILSGVGGWAGIGLAAGLTNPNDGLGAVGGYASLSQYTELGGQLTVFELPG

[0248] Exemplary methanol dehydrogenase (SEQ ID NO:31)

[0249]

[0250] Exemplary methanol dehydrogenase gene - peg.20 (SEQ ID NO:32)

[0251] In one embodiment, the vector comprises a gene encoding 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 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, 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.

[0252] MNRNTPKARGASSLAMAVAMGLAVLTTAPATANDQLVELAKDPANWVMTGRDYNAQNYSEMTDINKENVKQLRPAWSFSTGVLHGHEGTPLVVGDRMFIHTPFPNTTFALDLNEPGKILWQNKPKQNPTARTVACCDVVNRGLAYWPGDDQVKPLIFRTQLDGHIVAMDAETGETRWIMENSDIKVGSTLTIAPYVIKDLVLVGSSGAELGVRGYVTAYDVKSGEMRWRAFATGPDEELLLAEDFNAPNPHYGQKNLGLETWEGDAWKIGGGTNWGWYAYDPEVDLFYYGSGNPAPWNETMRPGDNKWTMAIWGREATTGEAKFAYQKTPHDEWDYAGVNVMMLSEQEDKQGQMRKLLTHPDRNGIVYTLDRTNGDLISADKMDDTVNWVKEVQLDTGLPVRDPEFGTRMDHKARDICPSAMGYHNQGHDSYDPERKVFMLGINHICMDWEPFMLPYRAGQFFVGATLTMYPGPKGDRQNALGLGQIKAYDAISGEMKWEKMERFSVWGGTMATAGGLTFYGTLDGFIKARDSDTGDLLWKFKLPSGVIGHPMTYKHDGRQYVAIMYGVGGWPGVGLVFDLADPTAGLGSVGAFKRLQEFTQMGGGVMVFSLDGESPYSDPNVGEYAPGEPT

[0253] Exemplary methanol dehydrogenase (SEQ ID NO:33)

[0254]

[0255] Exemplary methanol dehydrogenase gene - peg.3083 (SEQ ID NO:34)

[0256] An 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 with SEQ ID NO:35. In one embodiment, the vector comprises such a gene that comprises 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 with SEQ ID NO:36, or consists 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 with SEQ ID NO:36.

[0257] MTRSFDPDTLDLPRGHFIAGEHVADRGRLAMHRPSDGAAFGESPVADADMVDRAVAAGRAALAASGWGCGVPRDRTRALLKWADLIEAEAETLARFEAACSTRPVAQLPVGDIAVTAEQIRFFAEMADKEGSDLVPTRDASLGMTVDEPYGVVGAITPWNFPLSMAGWKLAPALAAGNAVVLKPSEMTPFSTLYMAELSVRAGIPAGLVNVVLGDGPVTGNAITGHPGIGKVSFTGSTGAGQAIMGNIARNGVKPMTLELGGKSPQIVFADADLDLAADCIARSITFNAGQACVAGSRVLVAAEIAEALAERLIARMADHRPGTTWDAETQYSPIISERQIARIDGIVQAAVAQGAEVLAGAARLDHPGWFYAPTLLAGVAPDSPAVTEEIFGPVLTLEPFADEEQAVAMADHPTYGLCAGIFTRDLSCALRVMRRIEAGTVWINRYGRSRDHILPTGGYKSSGIGKDLGRAAYHANRRQKSVLIDL

[0258] Exemplary aldehyde dehydrogenase (SEQ ID NO:35)

[0259]

[0260] Exemplary aldehyde dehydrogenase gene—peg.5153 (SEQ ID NO:36)

[0261] Alcohol dehydrogenase can convert 4-hydroxybutyric acid into succinic semialdehyde. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:37. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:38, or consists 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 with SEQ ID NO:38.

[0262] MDFTNLGVMRAPRHLVFGAGQRGALARHAGVFGTRALIVTDTRMARDKDFLQMRQALEAQGIATQVFDGVAAELPLSCIEAGAKAGRAAGARMIIGIGGGSCLDAAKIIGLLLSHGGAPQDYYGEYKVPGPIMPLILLPTTSGTGSEVTPVAVLDDPQRAMKIGIASPHLIPEIAICDPELTLSCPPGLTAASGADAMTHAIEAFTTLRRPADSGLSLDHVFIGKNAISDSLALEAIRLIAANLARCVSHGDDLEARSAMMLGSTLAGLAFGVAGTAAAHAIQYPVGAMTHTAHGLGVATLMPYVMAWNRPSCETDFARIGAAMGLAASGDTSRQAEAAIAAIAALFAQVGIPATIAQLGVPEDRLDEIARLALSAERLIKNNPRMLDAEGMDRIVRAAHSGDLDLLTATSPRKAALQ

[0263] Exemplary alcohol dehydrogenase (SEQ ID NO:37)

[0264]

[0265] Exemplary alcohol dehydrogenase gene - peg.245 (SEQ ID NO:38)

[0266] 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 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 with SEQ ID NO:39. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:40, or consists 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 with SEQ ID NO:40.

[0267] MSYHDTISQELGFDPQALRGDLWIDGGWRKGRGGDPIAVIDPSTGNTITRIENASIDDAMDAVAAAEAALPGWAATPPRVKSEILRRCYDLMIQRKDMLARLISLENGKALPDAQGEVLYAAEFFRWFAEEAVRLNGEIYTAPSGANRIIVTHRPIGVAVMVTPWNFPAAMATRKIAPALAAGCTCVLKPATETPLTAYALAEIYAEAGVPPGVVNVLTTSRSGATVSAMLHDPRVRKLSFTGSTEVGRRLLHEAADTVISCSMELGGNAPFIVFDDADLDLAIEGAMVAKMRNGGEACTAANRFLVQKGIAPAFAERLAARMEAMTLGAGYAGETLCGPLINREALDRIAGLVSEAESHGAKTLTGGRPLDRPGFYFPPTVLTDVPPQAEITGEEIFGPVAALATFETEDEAIARANSTEYGLISYVFTSDLARGLRVSERLDSGMVGLNRGVVSDPAAPFGGTKQSGLGREGAHHGILEFCEVQYIAANW

[0268] Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO:39)

[0269]

[0270] Exemplary succinic semialdehyde dehydrogenase gene—peg.246 (SEQ ID NO:40)

[0271] In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:41. In one embodiment, the vector comprises such a gene that comprises 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 with SEQ ID NO:42, or consists 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 with SEQ ID NO:42.

[0272] MTKHATDLKMLLKDPSLLQTRAYVAGEWVDADDGKTFPVVNPARGDVIAEVADLSRAEVARAIAAAAEAMKGWAARTAKGRAQIMRKWFDLMMENQDDLGRILTAEMGKPLPEAKGEIAYGASFIEWFGEEAKRIYGETIPGHLPDKRLTVIRQPIGVVGSITPWNFPNAMITRKCGPAIAAGCGFVGRPAAETPLSALALAVLAERAGIPKGLFSIVTSSRSSDIGKEFCENPLIRKLTFTGSTEVGRILLRQAADQVLKCSMELGGNAPFIVFDDADLDAAVEGAMASKFRNNGQTCVCANRIYVQAGVYDAFAQKLAAAVDKLRVGDGLEEGVTTGPLINQDAVEKVQEHIQDAVAGGATVVTGGKPREGLFFDPTVVTGITDKMKVATEETFGPLAPLFRFETEEEAVERANATIFGLASYFYARDIGRITRVQEALEYGIVGVNTGIISTEVAPFGGVKQSGLGREGSRHGIEDYLEMKYICLSI

[0273] Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO:41)

[0274]

[0275] Exemplary succinic semialdehyde dehydrogenase gene—peg.2316 (SEQ ID NO:42)

[0276] In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:43. In one embodiment, the vector comprises such a gene that comprises 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 with SEQ ID NO:44, or consists 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 with SEQ ID NO:44.

[0277] MTALKDKDLLRQQALIGGNWVDAASGAVVQVTDPATGQVMGTIPDLSAAETRAAIDAADAAFASWKKRSHAERAALLERWFDLMNQHAEDLALILTLEQGKPLSEARGEIAYGASFVKWFAEEARRIDGTVIPAPTNDRRILTLKEPVGVSAIITPWNFPNAMITRKVGPALAAGCTVVIKPSEFTPYSALALGVLAERAGIPAGVVNIVTGMPAEIGAELTANPTVRKVSFTGSTRVGSLLMAQSAPTVKRLSLELGGNAPFIVFDDADLDAAVEGAIASKFRNGGQTCVCSNRILVQAGVYDAFAEKLGAKVAAMKVGPGTQAGNDIGPMINRAALDKIARHVADAVAKGATVAARAEIPEGQYAAPVVLTGATTEMELASEETFGPVAPLFRFETEDEAVAIANGTPFGLAAYFYTENIRRAWRVAEALEFGMVGLNTGAVSTTVSPFGGVKSSGLGREGARAGIEEYLEVKAFHMGGL

[0278] Exemplary succinic semialdehyde dehydrogenase (SEQ ID NO:43)

[0279]

[0280] i PHA synthesis pathway

[0281] In one embodiment, the vector comprises one or more genes encoding all or part of the pathway for synthesizing PHA. Suitably, the vector comprises genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and / or PHA synthase.

[0282] 3-ketoacyl-CoA thiolase can catalyze the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:45. In one embodiment, the vector comprises a gene comprising 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 with SEQ ID NO:46, 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 with SEQ ID NO:46.

[0283] MSTDPIVIVGSARTPMGGFQGDFAGVEAAALGATAIKAALGGLDPQAVDEIIMGCVLPAGQGQAPARQAALGAGLPLGAGATTVNKMCGSGMKAAMLGHDLILAGSADVVVAGGMESMSNAPYLLPKARSGYRMGHGQVMDHMFLDGLEDAYDKGRLMGTFAEDCAEAYQFTREAQDEFAISSLTRAQKAIAAGHFTGEIAPVTVRGRGGETVVDTDEQPGKARPDKIPTLRPAFRKDGTVTAANSSSISDGAAALVLMRASEAERRGLVPRARILGHATFADKPGLFPTAPIGSVRRLLERTGTAIGDYDLFEVNEAFAVVAMAAMRDLGLSHDAVNVHGGACALGHPIGASGARVLVTLLAALETHGGRRGIASLCIGGGEATAVAIERMQ

[0284] Exemplary 3-ketoacyl-CoA thiolase (SEQ ID NO:45)

[0285]

[0286] Exemplary 3-ketoacyl-CoA thiolase gene - peg.2992 (SEQ ID NO:46)

[0287] In one embodiment, the vector comprises a gene encoding enoyl-CoA hydratase. Enoyl-CoA hydratase (EC 4.2.1.17) can also function as δ(3)-cis-δ(2)-trans-enoyl-CoA isomerase (EC 5.3.3.8), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and / or 3-hydroxybutyryl-CoA epimerase (EC 5.1.2.3), and can play a role in synthesizing hydroxybutyryl-CoA. In one embodiment, the vector comprises a gene encoding 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 with SEQ ID NO:47. In one embodiment, the vector comprises a gene that comprises 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 with SEQ ID NO:48, or consists 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 with SEQ ID NO:48.

[0288] MPVHYDLAGDSAVLTFDNPPLNVLGQAMRADLARAIAQAAADRPARLILRGAGRNFVAGADAREFDGPPLDPQLNEVLDALAALPFPTIAAIHGAALGGGLEIALACRFRIAHPSATLGLPEVTLGIVPGAGGTQRLPRLVGMAAALDLLGQGRSVTAAEAESLGLIDLIADDPMAAARGVDTQTLLRALCADDRPPPAPDEAAVAAAHARADRRAPGQVAPHRAIELVATSAQEPIKAALTRERATFLDLRGSDQARALRHVFFAERAAMAQGKAWPAPAPEIARAVVVGGGNMGAAIAYALLSAGLVVRVVETDAAALDRARDNIAGLVAQGRKRGALTDAGAAELQARLSLAVGYDDLPAADLAIEAAYEDMAVKQAIFAALQDALPDSTILATNTSYLDIDLLAQGIRQPGRFLGLHFFAPAHVMKLLEIVRGEATSDQTLGAAFRLARKLGKVPVLAGVCDGFIGNRILARYRHAADILLLEGALPAQVDAAMRGFGMAMGPYEAQDMSGLDIAYANRRRQNLRDRADHRYVPIADHLVERCRRLGRKSGAGWYDYDAEGRAQPSDEVTQAILSASRDAGITRVALPAEGIAERLVLAMIAEATRILAEGIAAAPRDIDLVLVHGYGFPRWRGGLMHHADRLTPARILSRIEALAKDDPLSWSVPPLLRQLADEGRDFTSLNPSA

[0289] Exemplary enoyl-CoA hydratase (SEQ ID NO:47)

[0290]

[0291] Exemplary enoyl-CoA hydratase gene—peg.203 (SEQ ID NO:48)

[0292] 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 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, 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.

[0293] MAGKDKKPEVEAGAGDAAKPRKARGTGAKARSAAKVAKQDPAPADSEAPARGRRAKAAPAAKAAQEPVTLAKPKAAAGSGAGKPAAEPVAKSGARKPVSRARAASRSDPRTGTRRKPSAKAAAKAQAEAASLSAVDEALRPLGAVGPGAAPPMAAPAAASPTPAGTERPAAPAAEPSSAAAFAEAAFGIGSRLPEQLAQNIERIESLTQRLISALAQRRPHSPGVELPGPELFATATGAWIKLLTEQPERVLSQQVSYWGETLRHFAEAQAALARGTLKPPPSEGLRDRRFSNPLWEAHPFFNFIKRQYQINAQALEEAASALDLPEMTDRRRIEWFTRQMIDMMAPTNFLATNPDALEKALETEGESLVKGLENLVRDVEQNNGELIVSLADRDAFRVGENIGTTEGTVVARTKLYELIQYKPTTAQVHEIPLVIFPPWINKFYILDLKPQNSLIKWIVDQGYTLFVVAWKNPDPSYGDTGMDDYVTAYLEVMDRVLDLTDQKKLNVVGYCIAGTTLALTLSILKQRGDDRVNSATFFTALTDFADQGEFTAYLQEDFVSGIEEEAARTGVLGAQLMTRTFSFLRANDLVWGPAIRSYMLGEMPPAFDLLFWNGDGTNLPGRMAVEYLRGLCQQNRFVKEGFDLLGHRLHVGDVTVPLCAIACETDHIAPWRDSWRGVAQMGSKDKTFILSESGHIAGIVNPPSKKKYGHYTSDAGFDQGEQHWLDKARHHEGSWWGRWGEWLARRAGNMVEARDPGEGFGPAPGLYVHERA

[0294] Exemplary PHA synthase (SEQ ID NO:49)

[0295]

[0296] Exemplary PHA synthase gene - peg.988 (SEQ ID NO:50)

[0297] e. Cells and kits

[0298] In one aspect, the present invention provides a cell comprising a vector according to the present invention. The cell can be an isolated cell.

[0299] The cell can be a microorganism, such as a bacterium, archaebacterium, 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 versutus. In some embodiments, the bacterium is Paracoccus denitrificans.

[0300] In one aspect, the present invention provides a kit for producing polyhydroxyalkanoate (PHA) from polyester waste.

[0301] The kit can comprise one or more microorganisms according to the present invention. The kit can comprise one or more vectors according to the present invention. The kit can comprise instructions for performing the methods of the present invention.

[0302] Examples

[0303] The present invention will now be further described by way of examples, which are intended to assist those skilled in the art in practicing the present invention and do not limit the scope of the present invention in any way.

[0304] f. Example 1: Generation of a genome-scale model (GSM)

[0305] The inventors have identified genetic traits that allow various biodegradable polyester wastes and their monomers to be metabolically converted separately by the same microorganism (e.g., Paracoccus denitrificans) to form a new biosynthesized polyester (PHA).

[0306] Gene traits were identified by generating a new genome-scale model (GSM) based on the genome of Paracoccus denitrificans PD1222 (see GenBank assembly accession number: GCA_000203895.1), which is publicly available. Paracoccus denitrificans PD1222 is a derivative of Paracoccus denitrificans DSM 413. The GSM was used for metabolic reconstruction by identifying genes in the genome that are previously described in different microorganisms for catabolizing each polyester component, as well as genes identified to produce the PHA polyester PHB. Using the GSM, we can not only uniquely identify the genes present in the genome, but also observe the possible metabolic network formation in the organism, thus identifying seemingly reasonable connections between the activities of different genes for metabolic outcomes. The target components are most of the monomers used industrially to produce the major plastic polyesters.

[0307] Table 1 shows the genetic traits and network connections identified in the genome related to the utilization of a large number of polyester monomers by Paracoccus denitrificans through aerobic or anaerobic metabolic pathways. 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 Visualization of the metabolic ability of Paracoccus denitrificans to convert polyester monomers based on the newly constructed GSM (genome-scale model)

[0308] The inventors first discovered that the pathway for 1,4-butanediol exists in this microorganism. Specifically, 1,4-butanediol is one of the constituent monomers of various polyesters and also has toxic effects on humans, so it is highly relevant to achieving biodegradation and recycling.

[0309] In the following examples, a derivative of Paracoccus denitrificans DSM 413 was used based on the newly constructed GSM.

[0310] Table 1. The summary table of the identified genes allows for the metabolic utilization of different polyester monomer components and the subsequent production of polyhydroxybutyrate (PHB) when present in the genome. These genes are part of the metabolic pathways identified in Paracoccus denitrificans PD1222 and are also present in other Paracoccus denitrificans including, for example: Paracoccus denitrificans ATCC 19367 (see Si, Y.Y. et al., 2019, Canadian journal of microbiology, Vol. 65, No. 7: pp. 486 - 495), Paracoccus denitrificans DYTN-1 (see Zhao, Y. et al., 2020, Letters in applied microbiology, Vol. 70, No. 4: pp. 263 - 273) and Paracoccus denitrificans R-1 (see Hu, C. et al., 2022, Microbiology Resource Announcements, Vol. 11, No. 4: pp. e01236 - 21).

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] g. Example 2: In vivo metabolism of polyester monomers and production of PHA under aerobic conditions

[0317] Each polyester monomer was separately tested in minimal medium as the sole carbon source to verify whether Paracoccus denitrificans could grow on this substrate and thus metabolize it. Additionally, tests were conducted to verify which monomer was consumed faster by Paracoccus denitrificans.

[0318] To test the ability to grow on substrates as the sole carbon source, batch cultures were carried out in mineral salts medium (MSM) and the corresponding monomers as the sole carbon source, under aerobic conditions and at 34 °C. The inoculum of 2% was from a culture in LB medium under the same conditions. The results in Table 2 confirmed that Paracoccus denitrificans could degrade the monomers of 12 different plastic polymers used, for example, in packaging.

[0319] Kinetic analysis was obtained in batch cultures carried out in a 2 L fermenter in mineral salts medium (MSM) and the corresponding monomers (0.3% weight / volume) as the sole carbon source, under aerobic conditions and at 37 °C. The inoculum was a 200 mL culture adapted to the same conditions with an OD 600 of approximately 0.1. The culture was carried out until maximum growth (maximum OD600 The presented data are the results of three replicates. Figure 3 shows the biomass formation (dry cell mass) of Paracoccus denitrificans when different monomers are supplied as the sole carbon source in a 0.3% (weight / volume) medium. Table 3 shows that even polyester monomers such as 1,4-butanediol, which are usually challenging for microbial processing, have a significant yield.

[0320] Table 2. Ability of Paracoccus denitrificans to grow on different monomers as the sole carbon source to verify its degradation and metabolism Metabolic potential of common plastic polymers .

[0321]

[0322] Table 3. Kinetic parameters for the assimilation of different monomers of plastic polymers by Paracoccus denitrificans as the sole carbon source when maximum growth is reached Figure 4 .

[0323]

[0324] The resulting microbial fermentation broth was analyzed by GC-MS, confirming the ability of Paracoccus denitrificans to produce PHA polymers from various polyester monomers used as substrates. Ratio % of different components 3HB or 3HV of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) And Table 4 shows that Paracoccus denitrificans produces polymers with different components of PHA polymers with different ratios according to the monomers used as substrates. The ratios of PHA species are also different.

[0325] Table 4. Biopolymer production by Paracoccus denitrificans. The yields (Y PHA ) relative to biomass and PHA polymerization are shown h. Example 3: In vivo metabolism of polyester monomers and production of PHA under anaerobic conditions .

[0326]

[0327]

[0328] Figure 5

[0329] Batch cultures were carried out under anaerobic conditions at 34 °C in mineral salt medium (MSM) and succinic acid (monomers of PBS, PBSA, PBST, PBSTIL) as the sole carbon source. A 2% inoculum was from a culture in LB medium under the same conditions. Table 5. Kinetic parameters for the assimilation of different monomers of plastic polymers by Paracoccus denitrificans as the sole carbon source when maximum growth is reached under anaerobic conditions And Table 5 shows that Paracoccus denitrificans is able to produce PHA using polyester monomers under anaerobic conditions.

[0330] i Example 4: In vivo metabolism of polyester polymers and production of PHA Figure 6 .

[0331]

[0332] Embodiments

[0333] Biopolymer recycling by Paracoccus denitrificans can also be achieved on polymers, not just on single monomers. In waste management, polymers (e.g., from packaging) reach entire waste treatment centers. Mechanical (e.g., shredding) and / or chemical treatment (e.g., alkali treatment) facilitate the degradation of complex compounds into smaller particles and make polymer monomers amenable to recycling. Biological recycling by Paracoccus denitrificans can also be achieved on pretreated polymers and produce new biopolymers (e.g., PHA).

[0334] The polymers were shredded (500 μm - 1000 μm) and chemically treated with an alkaline solution (2 M NaOH), incubated at 37 °C with continuous stirring (300 rpm) for 7 days. The polymers tested were PHB, PLA, PHBV, PHBH, PLA / PCL. The alkali-pretreated polymers were neutralized (neutralized to pH 7 with HCL) and 10% (v / v) was added to a mineral salt medium (90% (v / v)) as the sole carbon source. The flasks were inoculated with 1 mL of fresh Paracoccus denitrificans culture and incubated at 30 °C with rotary shaking at 300 rpm for 3 days. Biomass growth (estimated as optical density) and PHB content were monitored regularly.

[0335] ​ It was shown that the bacteria grew on all pretreated polymers as the sole carbon source and produced PHB. Up to 30% of the microbial cell mass of PHB was accumulated when using the hydrolysis products of PHB, PHBH or PHBV as substrates. When using PLA and PCL / PLA blends, 15% of PHB was accumulated. When using the hydrolysis products of PBS and PBAT, the accumulation was about 6%.

[0336] ​

[0337] The various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paragraphs).

[0338] 1. A method for producing polyhydroxyalkanoates (PHA) from polyester waste, the method comprising the steps of: (a) providing a culture medium containing polyester waste; and (b) culturing microorganisms in the culture medium to produce PHA, wherein the microorganisms utilize various polyester monomers from the polyester waste to produce the PHA.

[0339] 2. A method for producing polyhydroxyalkanoates (PHA) from polyester waste, the method comprising the steps of: (a) providing a culture medium containing polyester waste; and (b) culturing microorganisms in the culture medium to produce PHA, wherein the microorganisms utilize 1,4-butanediol from the polyester waste to produce the PHA.

[0340] 3. The method according to paragraph 1 or 2, wherein the microorganism is from the genus Paracoccus.

[0341] 4. The method according to any one of the preceding paragraphs, wherein the microorganism is Paracoccus denitrificans.

[0342] 5. The method according to any one of the preceding paragraphs, wherein the microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof.

[0343] 6. The method according to any one of the preceding paragraphs, wherein 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 CCM982, 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 IMET10380, Paracoccus denitrificans VKM B-1324, or Paracoccus denitrificans ICPB 3979.

[0344] 7. The method according to any one of the preceding paragraphs, 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 utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0345] 8. The method according to any one of the preceding paragraphs, wherein the microorganism comprises genes encoding each of the following: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxypentanoic acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0346] 9. A method according to any of the preceding paragraphs, wherein the polyester waste comprises 1,4-butanediol.

[0347] 10. A method according to any of the preceding paragraphs, 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 the group consisting of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol.

[0348] 11. A method according to any of the preceding paragraphs, wherein the polyester waste comprises succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol.

[0349] 12. A method according to any of the preceding paragraphs, wherein the polyester waste comprises the polyester monomers in free monomer form.

[0350] 13. A method according to any of the preceding paragraphs, 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 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), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxybutyrate (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0351] 14. A method according to any of the preceding paragraphs, wherein the polyester waste is pretreated, optionally wherein the polyester waste is mechanically treated and / or chemically treated.

[0352] 15. A method according to any of the preceding paragraphs, wherein the method further comprises the step of pretreating the polyester waste.

[0353] 16. A method according to any of the preceding paragraphs, wherein the method further comprises the step of mechanically treating the polyester waste.

[0354] 17. The method according to paragraph 16, wherein the polyester waste is shredded, optionally wherein 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.

[0355] 18. The method according to any one of the preceding paragraphs, wherein the method further comprises the step of chemically treating the polyester waste.

[0356] 19. The method according to any one of the preceding paragraphs, wherein the polyester waste is subjected to an alkali treatment.

[0357] 20. The method according to paragraph 19, wherein the polyester waste is neutralized after the alkali treatment.

[0358] 21. The method according to any one of the preceding paragraphs, wherein the culture medium 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.

[0359] 22. The method according to any one of the preceding paragraphs, wherein the culture medium comprises a mineral salt medium.

[0360] 23. The method according to any one of the preceding paragraphs, wherein the microorganism is cultured under aerobic or anoxic conditions.

[0361] 24. The method according to any one of the preceding paragraphs, wherein the microorganism is cultured under anoxic conditions.

[0362] 25. The method according to any one of the preceding paragraphs, wherein the microorganism is cultured for about one day to about seven days, about two days to about six days, or about three days to about five days.

[0363] 26. The method according to any one of the preceding paragraphs, wherein a single microbial strain is cultured.

[0364] 27. The method according to any one of the preceding paragraphs, wherein the method comprises a single culturing step.

[0365] 28. The method according to any one of the preceding paragraphs, 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.

[0366] 29. A method according to any of the preceding paragraphs, wherein the microorganism uses polyester monomers of a variety of polyesters from the polyester waste to produce the PHA, optionally wherein the microorganism uses polyester monomers of at least three, at least four, at least five, at least six, at least seven or at least eight polyesters from the polyester waste to produce the PHA.

[0367] 30. A method according to any of the preceding paragraphs, wherein at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 75 wt% or at least about 80 wt% of the polyester waste is utilized during the cultivation.

[0368] 31. A method according to any of the preceding paragraphs, 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.

[0369] 32. A method according to any of the preceding paragraphs, wherein at least about 10 μg of PHA / mg dry cell weight (DCW), at least about 20 μg of PHA / mg DCW, at least about 30 μg of PHA / mg DCW, at least about 40 μg of PHA / mg DCW, or at least about 50 μg of PHA / mg DCW is produced.

[0370] 33. A method according to any of the preceding paragraphs, wherein the PHA comprises polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof, or consists of polyhydroxybutyrate (PHB) or a copolymer thereof and / or polyhydroxyvalerate (PHV) or a copolymer thereof.

[0371] 34. A method according to any of the preceding paragraphs, wherein the PHA comprises polyhydroxybutyrate (PHB) or a copolymer thereof, or consists of polyhydroxybutyrate (PHB) or a copolymer thereof.

[0372] 35. A method according to any of the preceding paragraphs, wherein the PHA comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0373] 36. A method according to any of the preceding paragraphs, wherein the method further comprises the step of recovering the PHA.

[0374] 37. A culture solution comprising polyester waste and a microorganism, wherein the microorganism is capable of using polyester monomers of a variety of polyesters from the polyester waste to produce PHA.

[0375] 38. The culture medium according to paragraph 37, wherein the microorganism is as defined in any one of paragraphs 3 to 8, wherein the polyester waste is as defined in any one of paragraphs 9 to 14, and / or wherein the culture medium is as defined in paragraph 21 or 22.

[0376] 39. The culture medium according to paragraph 37 or 38, wherein the culture medium further comprises PHA, optionally wherein the PHA is as defined in any one of paragraphs 33 to 35.

[0377] 40. Polyhydroxyalkanoate (PHA), the PHA being produced by the method according to any one of paragraphs 1 to 39.

[0378] 41. An article, the article comprising the PHA according to paragraph 40 or consisting of the PHA according to paragraph 40.

[0379] 42. The article according to paragraph 41, wherein the article is a package.

[0380] 43. Use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism utilizes various polyester monomers from the polyester waste to produce the PHA.

[0381] 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 the PHA.

[0382] 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 wherein the polyester waste is as defined in any one of paragraphs 9 to 14.

[0383] 46. The use according to 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 the PHA.

[0384] 47. The use according to any one of paragraphs 43 to 46, wherein the microorganism utilizes polyester monomers of various polyesters from the polyester waste to produce the PHA, optionally wherein the microorganism utilizes polyester monomers of at least three, at least four, at least five, at least six, at least seven or at least eight polyesters from the polyester waste to produce the PHA.

[0385] 48. Use according to any one of paragraphs 43 to 47, wherein the PHA comprises polyhydroxybutyrate (PHB) or its copolymer and / or polyhydroxyvalerate (PHV) or its copolymer, or consists of polyhydroxybutyrate (PHB) or its copolymer and / or polyhydroxyvalerate (PHV) or its copolymer.

[0386] 49. Use according to any one of paragraphs 43 to 48, wherein the PHA comprises poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or consists of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).

[0387] 50. Use according to any one of paragraphs 43 to 49, wherein the microorganism produces the PHA under anoxic conditions.

[0388] 51. Use according to any one of paragraphs 43 to 50, wherein the microorganism produces the PHA in a single culture step.

[0389] 52. A microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, the microorganism comprising genes encoding a pathway for utilizing a plurality of polyester monomers and a pathway for synthesizing PHA, wherein the microorganism has been genetically engineered to express at least a part of one or more of the pathways.

[0390] 53. The microorganism according to paragraph 52, wherein the microorganism comprises genes encoding one or more, two or more, three or more, four or more, five or more, six or more, or seven or more pathways selected from the following: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxyvaleric acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0391] 54. The microorganism according to paragraph 52 or 53, wherein the microorganism comprises genes encoding the following: (i) a pathway for utilizing succinic acid; (ii) a pathway for utilizing lactic acid; (iii) a pathway for utilizing ethylene glycol; (iv) a pathway for utilizing adipic acid; (v) a pathway for utilizing 6-hydroxyhexanoic acid; (vi) a pathway for utilizing 3-hydroxybutyric acid; (vii) a pathway for utilizing 3-hydroxyvaleric acid; and (viii) a pathway for utilizing 1,4-butanediol.

[0392] 55. A microorganism according to any one of paragraphs 52 to 54, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing succinic acid, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding succinate dehydrogenase.

[0393] 56. A microorganism according to any one of paragraphs 52 to 55, wherein the microorganism has been genetically engineered to express a succinate dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0394] 57. A microorganism according to any one of paragraphs 52 to 56, wherein the microorganism has been genetically engineered to introduce a gene encoding succinate dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:2.

[0395] 58. A microorganism according to any one of paragraphs 52 to 57, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing lactic acid, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding D-lactate dehydrogenase.

[0396] 59. A microorganism according to any one of paragraphs 52 to 58, wherein the microorganism has been genetically engineered to express a D-lactate dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:3.

[0397] 60. A microorganism according to any one of paragraphs 52 to 59, wherein the microorganism has been genetically engineered to introduce a gene encoding D-lactate dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:4.

[0398] 61. A microorganism according to any one of paragraphs 52 to 60, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing ethylene glycol, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding alcohol dehydrogenase, aldehyde dehydrogenase, and / or glyoxylate reductase.

[0399] 62. A microorganism according to any one of paragraphs 52 to 61, wherein the microorganism has been genetically engineered to express one or more of the following: (i) an alcohol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:5; (ii) an aldehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:7; and (iii) a glyoxylate reductase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0400] 63. A microorganism according to any one of paragraphs 52 to 62, wherein the microorganism has been genetically engineered to introduce one or more of the following: (i) a gene encoding alcohol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 6; (ii) a gene encoding aldehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 8; (iii) a gene encoding glyoxylate reductase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 10.

[0401] 64. A microorganism according to any one of paragraphs 52 to 63, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing adipic acid, optionally wherein the microorganism has been genetically engineered to express 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.

[0402] 65. A microorganism according to any one of paragraphs 52 to 64, wherein the microorganism has been genetically engineered to express one or more of the following: (i) a long-chain fatty acid-CoA ligase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 11; (ii) an acyl-CoA dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 13; (iii) an enoyl-CoA hydratase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 15; (iv) a 3-hydroxybutyryl-CoA dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 17; and (v) a 3-oxoadipyl-CoA thiolase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 19.

[0403] 66. A microorganism according to any one of paragraphs 52 to 65, wherein the microorganism has been genetically engineered to introduce one or more of the following: (i) a gene encoding a long-chain fatty acid-CoA ligase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:12; (ii) a gene encoding an acyl-CoA dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:14; (iii) a gene encoding an enoyl-CoA hydratase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:16; (iv) a gene encoding a 3-hydroxybutyryl-CoA dehydrogenase and having at least 70% sequence identity with 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 with the nucleotide sequence of SEQ ID NO:20.

[0404] 67. A microorganism according to any one of paragraphs 52 to 66, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing 6-hydroxyhexanoic acid, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding an alcohol dehydrogenase and / or an aldehyde dehydrogenase.

[0405] 68. A microorganism according to any one of paragraphs 52 to 67, wherein the microorganism has been genetically engineered to express: (i) an alcohol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:21; and / or (ii) an aldehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:23.

[0406] 69. A microorganism according to any one of paragraphs 52 to 68, wherein the microorganism has been genetically engineered to introduce: (i) a gene encoding an alcohol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:22; and / or (ii) a gene encoding an aldehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:24.

[0407] 70. A microorganism according to any one of paragraphs 52 to 69, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing 3-hydroxybutyric acid, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding an acyl-CoA synthetase and / or a 3-hydroxybutyric acid dehydrogenase.

[0408] 71. A microorganism according to any one of paragraphs 52 to 70, wherein the microorganism has been genetically engineered to express: (i) an acyl-CoA synthetase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:25; and / or (ii) a 3-hydroxybutyrate dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:27.

[0409] 72. A microorganism according to any one of paragraphs 52 to 71, wherein the microorganism has been genetically engineered to introduce: (i) a gene encoding an acyl-CoA synthetase and having at least 70% sequence identity with 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 with the nucleotide sequence of SEQ ID NO:28.

[0410] 73. A microorganism according to any one of paragraphs 52 to 72, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing 3-hydroxyvalerate, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding an acyl-CoA synthetase, a 3-hydroxybutyrate dehydrogenase, and / or a 3-ketoacyl-CoA thiolase.

[0411] 74. A microorganism according to any one of paragraphs 52 to 73, wherein the microorganism has been genetically engineered to express one or more of the following: (i) an acyl-CoA synthetase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:25; (ii) a 3-hydroxybutyrate dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:27; and (iii) a 3-ketoacyl-CoA thiolase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO:29.

[0412] 75. A microorganism according to any one of paragraphs 52 to 74, wherein the microorganism has been genetically engineered to introduce one or more of the following: (i) a gene encoding an acyl-CoA synthetase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:26; (ii) a gene encoding a 3-hydroxybutyrate dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:28; (iii) a gene encoding a 3-ketoacyl-CoA thiolase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO:30.

[0413] 76. A microorganism according to any one of paragraphs 52 to 75, wherein the microorganism has been genetically engineered to express at least a part of a pathway for utilizing 1,4-butanediol, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding methanol dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase, and / or succinic semialdehyde dehydrogenase.

[0414] 77. A microorganism according to any one of paragraphs 52 to 76, wherein the microorganism has been genetically engineered to express one or more of the following: (i) a methanol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 31; (ii) a methanol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 33; (iii) an aldehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 23; (iv) an aldehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 35; (v) an alcohol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 37; (vi) an alcohol dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 5; (vii) a succinic semialdehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 39; (viii) a succinic semialdehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 41; and (ix) a succinic semialdehyde dehydrogenase aldehyde dehydrogenase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 43.

[0415] 78. A microorganism according to any one of paragraphs 52 to 77, wherein the microorganism has been genetically engineered to introduce one or more of the following: (i) a gene encoding methanol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 32; (ii) a gene encoding methanol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 34; (iii) a gene encoding aldehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 24; (iv) a gene encoding aldehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 36; (v) a gene encoding alcohol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 38; (vi) a gene encoding alcohol dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 6; (vii) a gene encoding succinic semialdehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 40; (viii) a gene encoding succinic semialdehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 42; and (ix) a gene encoding succinic semialdehyde dehydrogenase aldehyde dehydrogenase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 44.

[0416] 79. A microorganism according to any one of paragraphs 52 to 78, wherein the microorganism has been genetically engineered to express at least a portion of a pathway for the synthesis of PHA, optionally wherein the microorganism has been genetically engineered to express one or more genes encoding 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and / or PHA synthase.

[0417] 80. A microorganism according to any one of paragraphs 52 to 79, wherein the microorganism has been genetically engineered to express one or more of the following: (i) a 3-ketoacyl-CoA thiolase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 45; (ii) an enoyl-CoA hydratase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 47; and (iii) a PHA synthase having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 49.

[0418] 81. A microorganism according to any one of paragraphs 52 to 80, wherein the microorganism 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 with the nucleotide sequence of SEQ ID NO: 46; (ii) a gene encoding an enoyl-CoA hydratase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 48; (iii) a gene encoding a PHA synthase and having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 50.

[0419] 82. A microorganism according to any one of paragraphs 52 to 81, wherein the microorganism has been genetically engineered by transfection, by transduction or by gene editing.

[0420] 83. A microorganism according to any one of paragraphs 52 to 82, wherein the microorganism is a bacterium.

[0421] 84. A microorganism according to any one of paragraphs 52 to 83, wherein the microorganism is capable of producing PHA 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 selected from: succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol.

[0422] 85. A microorganism according to any one of paragraphs 52 to 84, wherein the microorganism is capable of producing PHA using each of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol.

[0423] 86. A vector comprising a gene encoding an enzyme having at least 70% sequence identity with any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 or 49.

[0424] 87. A vector, said vector comprising a gene, said gene comprising a nucleotide sequence having at least 70% sequence identity with any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 or consisting of a nucleotide sequence having at least 70% sequence identity with any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50.

[0425] 88. The vector according to paragraph 86 or 87, wherein said vector is a plasmid or a viral vector.

[0426] 89. A cell, said cell comprising the vector according to any one of paragraphs 86 to 88.

[0427] 90. The cell according to paragraph 89, wherein said cell is a microorganism, optionally wherein said cell is a bacterium.

[0428] Unless otherwise indicated, the practice of the present invention will employ conventional techniques, all of which are within the capabilities of those of ordinary skill in the art. Such techniques are described in the literature. It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing the attendant advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

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

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

3. The method according to claim 1 or 2, wherein the microorganism is Paracoccus denitrificans DSM 413 or a derivative thereof, optionally wherein 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, and Paracoccus denitrificans NCCB 22021.

4. The method according to any one of the preceding claims, 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 the group consisting of succinic acid, lactic acid, ethylene glycol, adipic acid, 6-hydroxyhexanoic acid, 3-hydroxybutyric acid, 3-hydroxypentanoic acid, and 1,4-butanediol.

5. The method according to any one of the preceding claims, wherein the polyester waste is pretreated, optionally wherein the polyester waste is mechanically treated and / or chemically treated.

6. The method according to any one of the preceding claims, wherein the microorganism is cultured under anoxic conditions, optionally wherein a single microorganism strain is cultured and / or wherein the method comprises a single culturing step.

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

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

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

10. Polyhydroxyalkanoate (PHA), wherein the PHA is produced by the method according to any one of claims 1 to 8.

11. An article, said article comprising the PHA according to claim 10, or consisting of the PHA according to claim 10.

12. Use of a microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, wherein the microorganism utilizes various polyester monomers from the polyester waste to produce the PHA.

13. A microorganism for producing polyhydroxyalkanoate (PHA) from polyester waste, said microorganism comprising genes encoding a pathway for utilizing various polyester monomers and a pathway for synthesizing PHA, wherein the microorganism has been genetically engineered to express at least a part of one or more of the pathways.

14. A vector, said vector comprising: a gene encoding an enzyme having at least 70% sequence identity with any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13、15、17、19、21、23、25、27、29、31、33、35、37、39、 41, 43, 45, 47 or 49; and / or a nucleotide sequence comprising or consisting of a nucleotide sequence having at least 70% sequence identity with any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50.

15. A cell, said cell comprising the vector according to claim 14, optionally wherein the cell is a microorganism, optionally wherein the cell is a bacterium.

16. A method for producing polyhydroxyalkanoate (PHA) from polyester waste, said method comprising:

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