In vitro bioproduction of specific chain length poly (hydroxyalkanoate) monomers

Abstract

An enzymatic method and system for producing homogeneous chain length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbons from long chain fatty acids or precursors thereof is disclosed. The method also produces specific homogeneous chain length polyhydroxyalkanoate monomers and / or polymers. Long chain fatty acids or precursors thereof can be contacted in vitro with an enzyme or a mixture of enzymes to produce homogeneous chain length polyhydroxyalkanoates.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 411,673, filed on September 30, 2022, which is expressly incorporated herein by reference in its entirety. Background Art

[0003] It is estimated that the production of petroleum-based polymers exceeds 300 million metric tons per year, and global production continues to increase. Most petroleum-based polymers are used to produce disposable products, such as plastic beverage bottles, straws, packaging, absorbent articles (including wearable absorbent articles) and other polymer wastes. Most of these plastic products are discarded and do not enter the recycling stream. In addition, the rapid increase of nanoplastics and microplastics (which are harmful to those organisms) found in organisms has greatly increased, causing further concerns. No matter what kind of plastic, it is becoming increasingly important to develop new polymers or methods to provide true recycling to mitigate the health and environmental impacts of petroleum-based plastics.

[0004] Biodegradable polymers (also referred to as "biopolymers") produced from renewable resources hold great promise in reducing the global accumulation of petroleum-based plastics in the environment. One class of such biopolymers is polyhydroxyalkanoates (PHAs). A great deal of work has been done on the family of small-chain PHAs, most notably polyhydroxybutyrate (PHB) polymers (including poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB)), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) and copolymers thereof. However, although PHAs exhibit thermoplastic properties similar to some petroleum-based polymers in some respects and thus may represent potential substitutes for petroleum-based polymers such as polypropylene and polyethylene, existing characterized PHAs have not yet inherently included all properties, such as processability, melting temperature, crystallinity, and connectivity, to form a one-to-one substitute for true petroleum-based polymers. For example, existing PHA synthesis, whether via synthesis or commercial purchase, is limited to C1-C8 PHAs, which has hindered research on PHA polymers comparable to polypropylene / polyethylene.

[0005] Polyhydroxyalkanoates are synthesized using multiple bacterial and archaeal genera including Halobacillus, Bacillus, Salinobacter, Flavobacterium, Chromohalobacter, Halomonas, Marinobacter, Vibrio, Pseudomonas, Halococcus, Halorhabdus, Haladaptatus, Natrialba, Haloterrigena, and Halorussus, making PHAs a viable alternative to petroleum-based polymers as they are fully biorenewable and biocompatible. Polyhydroxyalkanoates serve as energy reservoirs for these organisms. The production of polyhydroxyalkanoate polymers by the above-mentioned microorganisms involves a three-step enzymatic mechanism that starts with acetyl-CoA. In the formation of PHB, the first step is the catalysis of acetyl-CoA by PhaA (β-ketothiolase) to form β-ketoacyl-CoA. In turn, it is converted to R-3-hydroxyacyl-CoA by PhaB enzyme (β-ketoacyl-CoA reductase) in an NADP-dependent reaction. In the last step, R-3-hydroxyacyl-CoA is polymerized into PHB by PhaC (PHB synthase). In other words, the last step of the pathway involves the polymerization of hydroxyalkanoic acid monomers into polyhydroxyalkanoate polymers by polyhydroxyalkanoate polymerase. However, existing methods are only able to produce five straight-chain PHAs: hydroxypropionate, hydroxybutyrate, hydroxyvalerate, hydroxyhexanoate and hydroxyheptanoate, and purification is required if a single chain length is required. In addition, existing methods are generally unable to provide straight-chain and / or homogeneous PHA polymers.

[0006] Therefore, there is a need for systems and methods that can produce a wider range of chain length biopolymers in vitro. It would be another benefit if the biopolymers were suitable for use in consumer products and industrial processes. Additionally or alternatively, it would be economically and environmentally advantageous to produce homogeneous, linear and / or single polymer species, preferably without further purification or separation. It would be an additional benefit to provide an in vitro enzymatic method for forming such bioplastic polymers. Summary of the invention

[0007] Generally, the present disclosure relates to an enzymatic method for producing homogeneous chain length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbons. The method comprises contacting a long-chain fatty acid or a precursor thereof with an enzyme or enzyme mixture in vitro; and producing homogeneous chain length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbons.

[0008] In one aspect, the carbon chain length of the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers is equal to the central carbon chain length of the long chain fatty acid or its precursor. Additionally or alternatively, the central carbon chain length of the long chain fatty acid is any of the following carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30, and / or the central carbon chain length of the long chain fatty acid precursor is configured to produce a long chain fatty acid having any of the following central carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30. In addition, in one aspect, the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers are any one or more of the following carbon chain lengths: C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30.

[0009] In another aspect, the method is performed in one container, or alternatively, the method is performed in more than one container.

[0010] Additionally or alternatively, in one aspect, the long-chain fatty acid precursor is a long-chain alkane, a long-chain alcohol or a long-chain aldehyde. In addition, in one aspect, enzyme or enzyme mixture contains at least one enzyme selected based on the central chain length of long-chain fatty acid. In another aspect, the method comprises a second long-chain fatty acid or its precursor, the chain length of this second long-chain fatty acid or its precursor is different from long-chain fatty acid or its precursor, and the homogeneous chain length polyhydroxyalkanoate wherein produced is a copolymer with a long-chain fatty acid or its precursor and a segment of a second long-chain fatty acid or its precursor.

[0011] In addition, in one aspect, the method comprises one or more additional steps: long-chain fatty acids or its precursor are contacted with enzyme or enzyme mixture in vitro. In one aspect, the method comprises contacting long-chain fatty acids or its precursor with a first enzyme in vitro, then contacting with one or more subsequent enzymes in a stepwise manner. In another aspect, the method comprises contacting long-chain fatty acids or its precursor with two or more enzymes in vitro simultaneously. In one aspect, the enzyme or enzyme mixture is purified from an extremophilic microorganism, wherein in one aspect, the microorganism is a bacterium of the genus Halomonas, Lihuaxuella, Lysobacter, Alteromonas, Arthrobacter, Azospirillum, Empedobacter, Desulfovibrio, Halobacillus, Halobacteriovorax, Haloechinothrix, Halomarina, Halorussus, Haloterrigena, Isoptericola, Marinobacter In some embodiments, the microorganism is an engineered microorganism that has been genetically modified to secrete a specific enzyme and / or is at least one type of naturally occurring microorganism that naturally encodes a specific enzyme.

[0012] In one aspect, the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers have linear carbon chains. In another aspect, the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers are at least about 90% homogeneous.

[0013] Additionally or alternatively, in one aspect, the enzyme or enzyme mixture is a thermophilic enzyme. In one aspect, the thermophilic enzyme tolerates temperatures of about 40°C to about 120°C. In another aspect, the method is carried out at a temperature range of about 40°C to about 80°C. In addition, in one aspect, the method utilizes enzymes that effectively synergize in the same environment, which is characterized by the same or similar pH and temperature. In addition, in one aspect, the method is an organism-free method, wherein a long-chain fatty alcohol or a precursor thereof is contacted with an enzyme or enzyme mixture in an environment substantially free of any bacteria that secrete the same enzyme or the same enzyme mixture.

[0014] The present disclosure also generally relates to uncharacterized polyhydroxyalkanoates, which are polyhydroxyalkanoates having a carbon chain length greater than C8, wherein the polyhydroxyalkanoate is a linear polymer substantially absent any side chain pendant polymers.

[0015] The present disclosure also generally relates to a method for producing homogeneous chain-length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbons. The method comprises contacting a long-chain fatty acid with a long-chain fatty acid CoA ligase / synthetase to obtain a long-chain fatty acid acyl-CoA; contacting a long-chain fatty acid acyl-CoA with a long-chain acyl-CoA dehydrogenase, then with a long-chain-enoyl-CoA hydratase, then with a hydroxyacyl-CoA dehydrogenase or a long-chain-3-hydroxyacyl-CoA dehydrogenase to obtain a long-chain acetoacetyl-CoA; and contacting a long-chain acetoacetyl-CoA with an acetoacetyl-CoA reductase to obtain a hydroxyacyl-CoA for polymerization into a polyhydroxyalkanoate.

[0016] In one aspect, the long-chain fatty acid CoA ligase / synthetase is purified from Thermobifida halotoleran. Additionally or alternatively, in one aspect, the alcohol dehydrogenase is a fungal long-chain alcohol dehydrogenase purified from the group comprising the following fungal genera: Aureobasidium, Macroventuria, Lophium, Tothia, Trichodelitschia, Westerdykella, Didymosphaeria, Viridothelium, Delitschia, Zopfia, Myriangium, Rhizodiscina, Saccharata, Aaosphaeria, Amniculicola, Byssothecium, Aspergillus Aspergillus, Meira, Dissoconium, Lizonia, Aureobasidium, Morchella, Sodiomyces, Tilletiopsis, Jaminaea, Ceraceosorus, Testicularia, Tilletiopsis, Violaceomyces, Rhizopus, Alternaria, Hesseltinella, Neurospora, Ramularia, and Rhynchosporium.

[0017] Other features and aspects of the disclosure are discussed in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The disclosure that makes the present disclosure comprehensive and enabling is more particularly set forth in the remainder of the specification and with reference to the accompanying drawings, in which:

[0019] Figure 1 is a schematic diagram of potential starting molecule classes for the biosynthesis of specific PHAs;

[0020] Figure 2 is a schematic diagram of an enzyme reaction that produces a long-chain fatty acid CoA molecule that serves as a substrate for forming a specific chain length PHA;

[0021] Figure 3 is a schematic diagram of the enzymatic reaction for forming homogeneous PHA in vitro;

[0022] Figure 4is a graph showing the preferred distribution of substrate chain lengths of three halophilic thermospora long-chain fatty acid CoA synthetases;

[0023] FIG. 5A(i) to FIG. 5D(v) The sequences of various enzymes used in the Examples and Figures are shown;

[0024] Figure 6 is a graphical representation of the reduction in fatty acid concentration in the initial reaction of Example 1 (arachidic acid (C18), open circles; myristic acid (C12), solid circles);

[0025] Figure 7 is a diagram of the formation of pyrophosphate in the initial reaction of Example 1 (substrate: arachidic acid (C18), open circles; myristic acid (C12), solid circles); excitation wavelength 316 nm, emission wavelength 456 nm;

[0026] Figure 8 is a graphical representation of the formation of precipitated PHA of Example 1 (arachidic acid (C18), open circles; myristic acid (C12), closed circles);

[0027] Fig. 9 is a graphic representation of the depolymerization of the PHA formed of Example 1 (arachidic acid (C18), open circles; myristic acid (C12), closed circles) as measured by the decrease in optical density at 650 nm (due to scattering);

[0028] Fig.10 is a graphical representation of the formation of pyrophosphate in the initial reaction of Example 2 containing equal masses of arachidic acid (C18) and myristic acid (C12);

[0029] Fig.11 is a graphical representation of the formation of precipitated PHA of Example 2 containing equal masses of arachidic acid (C18) and myristic acid (C12) as measured by the increase in optical density at 650 nm (due to scattering);

[0030] Fig.12 is a graphical representation of the depolymerization of the formed PHA of Example 2 containing equal masses of arachidic acid (C18) and myristic acid (C12) as measured by the decrease in optical density at 650 nm (due to scattering);

[0031] Fig.13 The method of Example 3 using hexacosanoic acid (CH3(CH2) 24 Schematic representation of pyrophosphate formation in the initial reaction of 2-nitropropene (COOH) as the sole substrate;

[0032] Fig.14As measured by the increase in optical density (due to scattering) at 650 nm, Example 3 using hexacosanoic acid (CH3(CH2) 24 Schematic representation of the formation of precipitated PHA using COOH) as the sole substrate;

[0033] Fig.15 As measured by the decrease in optical density (due to scattering) at 650 nm, Example 3 using hexacosanoic acid (CH3(CH2) 24 Schematic representation of the depolymerization of PHA formed by using 2-(4-(2-hydroxy-1-oxo-4-oxo-1-yl)-4-nitropropene (COOH) as the sole substrate (PHA depolymerase from T. thermophilus, filled circles; L. thermophila, open circles);

[0034] Fig.16 is a graphical representation of the formation of pyrophosphate in the initial reaction of Example 4 using heptanoic acid (CH3(CH2)5COOH) as the sole substrate;

[0035] Fig.17 is a graphical representation of the formation of precipitated PHA of Example 4 (the reaction was started with heptanoic acid (CH3(CH2)5COOH) as the sole substrate) as measured by the increase in optical density (due to scattering) at 650 nm;

[0036] Fig.18 is a graphic representation of the depolymerization of the PHA formed in Example 4 (the reaction was started with heptanoic acid (CH3(CH2)5COOH) as the sole substrate, the PHA depolymerase was from Xulihua thermophila);

[0037] Fig.19 is a consensus maximum likelihood tree showing the evolutionary relationships among the five most unique fatty acid-CoA ligases expressed by Halomonas titanicae ;

[0038] Fig. 20 is a graphical representation of the reduction in fatty acid concentration in the initial reaction of Example 5 (octanoic acid (C6), solid circles; myristic acid (C12), solid squares; behenic acid (C20), open circles);

[0039] Fig.21 is a diagram of the formation of pyrophosphate in the initial reaction of Example 5 (substrates: octanoic acid (C6), solid circles; myristic acid (C12), solid squares; behenic acid (C20), open circles); excitation wavelength 316 nm, emission wavelength 456 nm;

[0040] Fig. 22is a graphical representation of the formation of the precipitated poly(hydroxyalkanoate) of Example 5 (octanoic acid (C6), filled circles; myristic acid (C12), filled squares; behenic acid (C20), open circles); and

[0041] Fig.23 is a graphical representation of the depolymerization of the poly(hydroxyalkanoate) formed of Example 5 (octanoic acid (C6), filled circles; myristic acid (C12), filled squares; behenic acid (C20), open circles) as measured by the decrease in optical density at 650 nm (due to scattering).

[0042] Repeated use of reference characters in the present specification and drawings is intended to represent same or analogous features or elements of the invention. DETAILED DESCRIPTION

[0043] definition

[0044] The terms "about", "approximately" or "substantially" are used herein to modify a value to indicate that the value can be increased or decreased by 10% (such as 7.5%, such as 5%, such as 4%, such as 3%, such as 2% or such as 1%) and remain within the disclosed aspects. In addition, the term "substantially free of" is not limited to being completely or completely free of when used to describe the amount of a substance in a material, and can be equivalent to the lack of any perceptible or detectable amount of the listed substance in the material. Thus, for example, when the amount of a substance in a material is less than the precision of an industry-recognized instrument or test for measuring the amount of a substance in a material, the material is "substantially free of" the above-mentioned substance. In certain aspects, when the amount of a substance in a material is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% or less than 0.1% by weight of the material, the material can be "substantially free of" the above-mentioned substance.

[0045] As used herein, the term "biodegradable" or "biodegradable polymer" generally refers to a material that can be degraded by the action of naturally occurring microorganisms (such as bacteria, fungi, archaea, and algae); ambient heat; moisture; or other environmental factors. The biodegradability of a material can be determined using ASTM Test Method 5338.92.

[0046] As used herein, the term "enzyme" generally refers to enzymes including, but not limited to, native enzymes, purified enzymes, wild-type enzymes, modified enzymes, or combinations thereof.

[0047] As used herein, the term "microorganism" includes wild-type or modified bacteria, fungi, archaea, and algae that express or produce one or more of the enzymes discussed herein.

[0048] As used herein, the term "polyhydroxyalkanoate" or "hydroxyalkanoate" generally refers to a chemical family of biopolymers that includes, but is not limited to, the following members: polyhydroxybutyrate (PHB) polymers, including poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), and uncharacterized PHAs with carbon chains greater than C8 (as discussed in detail below), as well as monomers and copolymers of each of them.

[0049] Detailed Description

[0050] Those skilled in the art will appreciate that this discussion is merely a description of exemplary aspects and is not intended to limit the broader aspects of the present disclosure.

[0051] In general, the present disclosure relates to an enzymatic method for producing homogeneous poly(hydroxyalkanoates) (PHAs) in a manner that produces polymers of a single molecular species. That is, the present disclosure has surprisingly discovered that the unique in vitro synthetic route discussed herein allows for the production of a single polymer species without the need to purify the produced PHA polymer pool to achieve length homogeneity. In addition, the in vitro method discussed herein is also surprisingly capable of producing new PHAs that have not been described or failed to be produced by other methods, such as PHAs with chain lengths greater than eight carbons. That is, without wishing to be bound by theory, the present disclosure has surprisingly discovered that by carefully selecting enzyme combinations and process conditions, new PHA polymers of homogeneous but various chain lengths (e.g., carbon chain lengths greater than those currently thought possible) can be produced without the need for multi-step or multi-container methods, and even avoiding in vivo methods that limit the production of various different PHAs.

[0052] That is, the present disclosure has surprisingly discovered that starting materials of specific chain lengths (including long chains) can be enzymatically converted into acetyl-CoA precursors, which can then be subsequently enzymatically converted into PHAs of the corresponding specific chain lengths / long chain lengths. For example, the in vitro method according to the present disclosure utilizes a long-chain substrate material for in vitro enzymatic conversion, ensuring that the resulting PHA polymer is completely homogeneous. In addition, using the methods discussed herein, PHA long-chain copolymers can be produced by starting from two or more different chain length substrates. In such an aspect, the final PHA copolymer will reflect the composition and ratio of the input substrate molecules. Therefore, the method of the present disclosure provides an alternative to existing biological methods, which results in the formation of mixed-size PHA classes and the ability to produce longer PHA polymers, rather than short-chain to medium-chain polymers obtained by current in vivo bioreactor methods.

[0053] In one aspect, the starting material for the methods as discussed herein can be any long chain fatty acid or a single chain length precursor thereof. For example, a long chain fatty acid can contain a C6-C30 carbon chain (excluding the carbon at the carboxyl terminal end). (This carbon chain length can also be referred to as a long chain fatty acid n-2 carbon to account for the cleaved CH2COOH, which will be discussed in more detail below). Figure 1 As shown, in one aspect, the long chain fatty acids themselves can be suitable precursors.

[0054] However, depending on the available precursor sources, aldehyde, alcohol or alkane precursors of long-chain fatty acids can also be utilized. Figure 1 As will be appreciated in the art, and as a specific example only, the conversion from long-chain alkanes to long-chain alcohols can be performed by alkane monooxygenases (such as, EC 1.14.15.3, by way of example only); the formation of long-chain aldehydes from long-chain alcohols can be catalyzed by alkane-1-ol dehydrogenases / oxidoreductases (such as EC 1.1.99.20, by way of example only) or by long-chain alcohol dehydrogenases (such as EC 1.1.1.2, by way of example only); and the formation of long-chain fatty acids from aldehydes can be catalyzed by aldehyde dehydrogenases (such as EC 1.2.5.2, by way of example only). As shown, when starting with long-chain fatty acids, no enzymatic conversion is required to enter the PHA synthesis cycle. However, as described, even when starting with alkane precursors (such as, alkanes obtained from post-consumer and / or recycled sources, in only one aspect), the methods discussed herein can also be fully enzymatic methods. Furthermore, as will be discussed in more detail below, even starting from alkanes, alcohols, or aldehydes, careful selection of chain lengths and careful choice of enzymes that can coexist without negative interactions or side reactions can allow for a "one-pot" reaction.

[0055] However, if Figure 2 As shown, after obtaining or producing a long-chain fatty acid with a desired chain length n, it is converted into a coenzyme A moiety. In one aspect, the conversion into a coenzyme A moiety is performed by a long-chain fatty acid coenzyme A synthetase (also referred to in the art as a ligase or synthetase, such as EC 6.2.1.3, by way of example only). The reaction and the specific cofactors of the reaction require Figure 2 As shown in FIG. 1 . Furthermore, as is known in the art, in nature, the formation of PHB begins with the formation of acetoacetyl-CoA. Figure 2 As shown, the methods of the present disclosure allow for the formation of PHA starting from the formation of long-chain fatty acid acyl-CoA instead. n , where n is the chain length of the fatty acid. Thus, it allows for the accurate and efficient formation of long-chain and homogeneous PHAs.

[0056] Then, Figure 3A four-step method is shown in more detail in , which converts long-chain fatty acid CoA into a form that can be polymerized by PHA polymerase (which may also be referred to as synthetase, synthase or ligase). The first reaction is that of a long-chain acyl-CoA dehydrogenase (such as EC 1.3.8.8, by way of example only), which allows the production of long-chain trans-2,3-dehydroacyl-CoA. This in turn is the substrate for the second reaction, which uses a long-chain-enoyl-CoA hydratase (such as EC 4.2.1.17, by way of example only), to form long-chain hydroxyacyl-CoA. The third reaction forms a long-chain oxoacyl-CoA (using a hydroxyacyl-CoA dehydrogenase, such as EC 1.1.1.35, by way of example only), or alternatively uses a specific long-chain-3-hydroxyacyl-CoA dehydrogenase (such as EC 1.1.1.211, by way of example only). Finally, long-chain hydroxyacyl-CoA is formed by acetoacetyl-CoA reductase (such as EC 1.1.1.36, by way of example only).

[0057] Once the long-chain hydroxyacyl-CoA is formed, the long-chain hydroxyacyl-CoA is polymerized by a PHA polymerase (such as 2.3.1.34, just as an example) to form a specific homogeneous PHA monomer of chain length n. Figure 3 As shown, the PHA n formed will be a multiple of the chain length n of the monomer long chain -3-hydroxyacyl-CoA. For example, if the entire reaction is run with a C12 fatty acid, the length n of the resulting PHA n will be a multiple of 12 (e.g., for each polymerization event, n = 24, 36, 48, etc.). Of course, as discussed above, in an additional aspect, the polymer can be a copolymer of two specific input monomers. For example, if n = C12 and b = C18 fatty acids (or their precursors) are used as input, the resulting PHA polymer will be a copolymer of C12 and C18 monomer species.

[0058] While it should be understood that, in one aspect, enzymatic methods can be performed using enzymes from a single source, it should also be clear that the methods discussed herein provide additional benefits over in vivo methods, namely, enzymes purified from different sources can be combined based on input and kinetics, and / or multiple forms of a single enzyme can be utilized. For example, by utilizing the methods disclosed herein, the best enzyme can be used for each step in the method, even if the initial source is from a completely different bacteria. That is, the methods discussed herein are very capable of producing specific PHAs without the occurrence of additional metabolic side reactions that are typically present in in vivo methods, and without consuming metabolic energy on growth and cell division, thereby improving efficiency. In addition, by utilizing the enzymatic methods disclosed herein, any or all enzymes can be engineered to increase properties that make the enzyme more compatible with the overall process, such as site-directed mutagenesis to change stability, kinetic rate, etc., or a combination thereof.

[0059] For example, in one aspect, two or more enzymes purified from a single source may exhibit preferences for reactions with completely different chain lengths, particularly as described above with respect to Figure 3 The long-chain fatty acid CoA synthetase in question. That is, reference Figure 4 , three long-chain fatty acid CoA synthetases from halophilic thermosporidial fungi were purified. As shown, each enzyme has a preference for a different central chain length (wherein the carbon chain length refers to the central part of the molecule as described above, e.g., C14 refers to CH3(CH2) 14 COOH. WP_068688876: cross-labeled bars, WP_068692787: black bars, WP_06869753.1: shaded bars). Thus, in one aspect, the methods as discussed herein include enzymes from two or more organisms to improve stability, kinetic rate or other performance factors based on the carbon chain length of the precursor.

[0060] In addition, as will be discussed in more detail below, in one aspect, thermophilic enzymes can be utilized because, in just one exemplary aspect, reacting at higher temperatures can increase the thermodynamic driving force. However, as described above, enzymes from any source can be used as long as they catalyze the reaction of a certain length of fatty acid carbon chain.

[0061] However, in one aspect, the present disclosure generally relates to contacting one or more homogeneous long-chain fatty acid monomers (chain length (excluding the carboxyl terminus) of C8 or greater) or their alkane, alcohol or aldehyde precursors with an enzyme or enzyme mixture in vitro, and producing one or more C8 or greater PHA bioplastic monomers or polymers. In one aspect, the long-chain fatty acid monomers (chain length (excluding the carboxyl terminus) of C8 or greater or their alkane, alcohol or aldehyde precursors can be obtained from post-consumer materials, such as post-consumer personal care products, food industry products, packaging, post-consumer medical products, post-consumer industrial products, and other articles. Post-consumer products can also include components and / or other polymer wastes after industrial use.

[0062] In one aspect, the product may contain pollutants after use. The methods and systems herein can eliminate or reduce such pollutants. The product may contain pollutants after use, including but not limited to mesophilic pathogens, such as but not limited to viruses, bacteria, fungi and protozoa, which pollutants can eliminate pathogenicity by the disclosed method. As used herein, the term "mesophile" ("mesophile" and "mesophilic") refers to organisms naturally present in environmental conditions where humans usually coexist with organisms, and the environmental conditions include close to human body temperature (e.g., about 20°C to about 45°C), the salt content in water is about 5 to about 18 parts per thousand (also referred to as medium salt), about one atmosphere (e.g., about 20kPa to about 110kPa) and close to neutral pH (e.g., about pH 5 to about pH 8.5, also referred to as neutrophil ("neutrophile" or "neutrophilic")). Typical bacterial pathogens contemplated herein may include those commonly found in human feces, such as, but not limited to, Streptococcus, Bifidobacterum, Lactobacillus, Staphylococcus, Clostridium, Enterobacteriaceae, or Bacteroides.

[0063] However, it should be understood that in one aspect, the precursors can be obtained from non-recycled or non-post-consumer products, or can be produced specifically for the methods as described herein. For example, in one aspect, one or more precursors can be formed by a method involving forming a precursor.

[0064] However, regardless of the source of the precursor, the present disclosure generally relates to combining an enzyme or enzyme mixture (particularly selected to perform one or more reactions suitable for converting long-chain fatty acid monomers into homogeneous long-chain PHA monomers) with a long-chain fatty acid monomer or its alkane, alcohol or aldehyde precursor having a chain length (excluding the carboxyl terminus) of C8 or greater. In addition, it is believed that this mechanism can avoid the production of microplastics and nanoplastics, which is an additional benefit compared to non-bio-based polymers, because microplastics and nanoplastics are increasingly being concerned because they may have higher toxicity than intact petroleum-based polymers.

[0065] Furthermore, as described above, in one aspect, Figure 1 (as needed, if not starting with long-chain fatty acids) to Figure 3 The reactions can be run together or separately depending on the design of the overall process or by careful selection of enzymes. That is, as described above, and not wishing to be bound by theory, the present disclosure has found that by carefully selecting enzymes that are required (or flourish) under similar conditions and do not utilize Figures 1 to 3 The methods discussed herein starting with long-chain fatty acids or their precursors can be performed as a "one-pot" reaction, allowing for further improvements in efficiency, rate, and footprint.

[0066] However, in one aspect of the methods disclosed herein, the long chain fatty acid monomers having a chain length (excluding the carboxyl terminus) of C8 or greater, or their alkane, alcohol or aldehyde precursors, have any one or more of the following carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30. (Wherein, when starting from a fatty acid precursor, the chain length as discussed above is a chain length sufficient to produce a central chain length (excluding the carboxyl terminus) in the long chain fatty acid monomer that is the same as the length of the precursor). Exemplary fatty acids include arachidic acid (C18 central chain), myristic acid (C12 central chain) and hexacosanoic acid (C24 central chain) according to the above list. However, it should be understood that the fatty acid may be a fatty acid having a C6 to C30 central chain as discussed above.

[0067] In another aspect, the method of the present disclosure can be performed at a temperature range of about 40° C. to about 80° C., such as about 45° C. to about 75° C., about 50° C. to about 70° C., or about 55° C. to about 65° C. For example, the method can be performed at a temperature of about 40° C., about 45° C., about 50° C., about 60° C., about 65° C., about 70° C., about 75° C., and / or about 80° C. In one aspect, the method of the present disclosure can be performed in one container or in more than one container together.

[0068] In another aspect, as described above, it has been surprisingly found that the method can be fine-tuned by careful identification and selection of enzymes to suit specific reactions in industrial and / or laboratory-scale methods. For example, this can include careful calibration of reaction conditions, such as, for example, enzyme catalytic efficiency, optimal pH, or substrate discrimination. It can also include careful calibration of the overall reaction environment, such as, for example, selection of high temperature, high salt conditions, increased reaction pressure, and / or extreme pH or low temperature. For example, if the reaction conditions are such that the temperature is increased, a thermophilic enzyme can be selected. In addition, if a polyextremophilic enzyme is utilized, a variety of extreme conditions can also be used to carry out certain reactions of the present disclosure. In one aspect, thermophilic or thermostable enzymes can be utilized to produce PHA from long-chain fatty acids. In particular, thermophilic enzymes can be very suitable due to the favorable thermodynamics of catalysis at high temperatures, however, based on careful selection taking into account subsequent reactions and various reaction products, catalysis such as Figure 1-3 Any source of enzyme in the reaction.

[0069] Thus, in one aspect, the method may include a step of contacting the long chain fatty acid or its precursor with an enzyme or enzyme mixture in vitro. That is, without wishing to be bound by theory, the present disclosure has found that by carefully selecting enzymes that are required (or flourished) under similar conditions and that are not utilized in Figures 1 to 3 The methods discussed herein starting with long-chain fatty acids or their precursors can be performed as a "one-pot" reaction, allowing for further improvements in efficiency, rate, and footprint. However, in one aspect, the method can include one or more additional steps: for the above-discussed and Figures 1 to 3 Each in the one or more other steps shown in, by repeatedly introducing one or more enzymes, long-chain fatty acids or its precursor are contacted with enzyme or enzyme mixture in vitro. For example, one or more other steps can be repeated more than twice, more than three times, more than four times, more than five times, more than six times, more than seven times, more than eight times, more than nine times or more than ten times. For example, one or more other steps can be repeated less than three times, less than four times, less than five times, less than six times, less than seven times, less than eight times, less than nine times or less than ten times.

[0070] In addition, in another aspect, the method can include contacting the long-chain fatty acid or its precursor with the first enzyme in vitro, and then contacting one or more subsequent enzymes in a stepwise manner. For example, the long-chain fatty acid or its precursor can be contacted with the first enzyme in vitro, and then can be contacted with the second enzyme, and then contacted with one or more optional subsequent enzymes. In another aspect, the method can include contacting the long-chain fatty acid or its precursor with two or more enzymes in vitro simultaneously.

[0071] In one aspect, the first enzyme, the second enzyme and / or one or more subsequent enzymes can be different from each other. In another aspect, the first enzyme, the second enzyme and / or one or more subsequent enzymes can comprise some enzymes different from each other, and some enzymes can be identical or similar. For example, one or more subsequent enzymes can comprise at least one other enzyme different from the first enzyme.

[0072] For example, the sequences of enzymes that can be utilized in the methods of the present disclosure are shown in Figure 5. Although only exemplary enzymes are shown, the present disclosure has discovered that by carefully calibrating the reaction and environmental conditions and thoughtfully selecting enzymes that catalyze specific reactions, the methods of the present disclosure can produce homogeneous PHA monomers and polymers of specific lengths. For example, engineered enzyme variants can also be adapted to specific reactions or overall reaction processes.

[0073] In one aspect, the enzyme or enzyme mixture can be purified from an extremophilic microorganism. For example, the microorganism can be a bacterium of the genus Halomonas, Lihuaxuella, Lysobacter, Alteromonas, Arthrobacter, Azospirillum, Empedobacter, Desulfovibrio, Halobacillus, Halobacteriovorax, Haloechinothrix, Halomarina, Halorussus, Haloterrigena, Isoptericola, Marinobacter , Methyloligella, Micromonospora, Natronococcus, Nocardiopsis, Paracoccus, Roseivax, Saccharomonospora, Shewanella, Alicyclobacillus, Natranaerobius, Halobacteriaceae, Hyphomonas, Amycolatopsis, Georgenia, Acidothermus, Thermobifida, or a combination thereof.

[0074] In one aspect, the enzyme or enzyme mixture may include a thermophilic enzyme. For example, the thermophilic enzyme can tolerate temperatures of about 40°C to about 120°C, such as about 50°C to about 110°C, about 60°C to about 100°C, or about 70°C to about 90°C. Thus, in some aspects, the extremophilic enzyme used in the disclosed methods and systems may be an enzyme that exhibits a T optA thermophilic enzyme at about 40°C or more, about 50°C or more, about 60°C or more, about 70°C or more, about 80°C or more, or about 90°C or more. Exemplary thermophiles contemplated herein (and thermophilic enzymes produced therefrom) can include, but are not limited to: Alicyclobacillus pomorum (WP-084453829), Amycolatopsis thermoflava (WP-123687648), Amycolatopsis thermalba (WP-094002797), Amycolatopsis rumanii (WP-116109633), Azospirillum thermophilum (WP-109324320), Deinococcus actinosclerus (WP-082689076), Fervidobacterium gondwanense (SHN54810), Gandjariella thermophila ... thermophila)(WP-137812779), Georgenia satyanarayanai(WP-146237554), Hyphomanas sp.(HAO37884), Lihuaxuella thermophila(WP-089972404), Microbulbifer thermotolerans(P-197462976), Minwuia thermotolerans(WP-206420073), Rhodopseudomonas thermotolerans(WP-114356866), Rhodopseudomonas pentothenatexigens(WP-114356866), Streptomyces thermophilus thermovulgaris)(WP-067396676), Thermanaeromonas toyohensis(WP-084666479), Thermoactinomyces sp.)CICC 10523 (WP-198056464), Thermoactinomyces daqus (WP-033100012), Thermoactinomyces spp.)(NUT44302), Thermoactinospora rubra (WP-084965756), Thermobifida halotolerans (WP-068692693), Thermobifidafusca (WP-011290529), Thermobispora bispora (WP-206206594), Thermocatellispora tengchongensis (WP-185055796), Thermochromatium tepidum)(WP-153975900), Thermocrispummunicipal(WP-028851041), Thermoflavimicrobiumdichotomicum(WP-093229000), Thermogemmatisporacarboxidivorans(WP-081839208), Thermogemmatisporaaurantia(WP-151728970), Thermogemmatispora tikiterensis(WP-11243376), Thermogemmatispora onikobensis(WP-084659191), Thermoleophilaceae(MBA2429278), Thermomonospora echinospora) (WP-160147065), Thermomonospora cellulosilytica (WP-182704610), Thermomonospora amylolytica (WP-198679325), Thermostaphylospora chromogena (WP-093263254), Thermus thermophilus (WP-197735236), Thermus aquaticus (WP-053768217), Thermus islandicus (HEO42284). For example, at least one enzyme in step c) may be purified from thermophilic Xulihua bacteria. .

[0075] In another aspect, microorganisms from which enzymes can be purified can be selected based on factors including, but not limited to, growing easily and rapidly at high density, not requiring special media, being aerobic, having fast kinetics, being stable, being tolerant of high salt environments, being tolerant of temperature environments, being able to produce easily purified enzymes, lacking unconventional isoelectric points, not requiring enhanced biosafety measures, not containing excess cysteine ​​residues, being generally non-in vivo, being commercially available, or a combination thereof.

[0076] However, although enzymes present in solution have been discussed so far, it will be appreciated that in one aspect, the method can be carried out using one or more microorganisms that naturally express the enzyme in question or have been modified to express the desired enzyme. For example, the microorganism can be at least one type of naturally occurring microorganism that naturally encodes a particular enzyme for the method discussed herein.

[0077] In addition to the microorganism of natural expression of a certain enzyme gene, one or more genetically modified bacteria can also be selected, and the bacterial expression can carry out the exogenous enzyme of the specific reaction of the present invention. In addition, the microorganism can be an engineered microorganism, and this engineered microorganism has been genetically modified to secrete the specific enzyme for this paper method.

[0078] For example, any genus of bacteria or archaea can be matched with any enzyme expressed from a constitutive vector coupled to the correct signal sequence. In this regard, any suitable Gram-positive or Gram-negative bacteria can be used to produce and secrete the enzyme of interest. In this way, the enzyme can be customized based on environmental variables, the type and amount of the product after use, or a combination thereof. In addition, the sequence of the enzyme can be matched to the environment by selecting one from a sequence that is known (e.g., NCBI database) or with a fully or partially engineered variant. In one aspect, the selected bacteria or archaea can be transformed with a plasmid vector having a gene encoding a constitutively expressed specific enzyme (containing an appropriate signal sequence). Alternatively, the selected bacteria and / or archaea can be inserted into the bacterial chromosome by transduction, linear recombination, or any other suitable method rather than using an additional chromosomal vector, thereby eliminating the need for an exogenous vector.

[0079] Enzymes can be expressed by transforming suitable host organisms, for example, by using prokaryotic or eukaryotic host cells. Examples of host cell types include, but are not limited to, bacterial cells (e.g., Escherichia coli), yeast cells (e.g., Pichia pastoris, Saccharomyces cerevisiae), cultivated insect cell lines (e.g., Drosophila), plant cell lines (e.g., corn, tobacco, rice, sugarcane, potato tubers), mammalian cell lines (e.g., Chinese hamster ovary (CHO)). In one aspect, a recombinant host cell system can be selected that processes and post-translationally modifies the nascent polypeptide in a manner required to produce the final catalytic enzyme.

[0080] The nucleic acid sequence encoding the enzyme can be placed in an expression vector for expression in a selected host. Such expression vectors can generally include a transcription initiation region connected to the nucleic acid sequence encoding the enzyme. The expression vector can also include a plurality of restriction sites for inserting nucleic acid to be under the transcriptional regulation of various control elements. The expression vector can additionally contain a selective marker gene. Suitable control elements (such as enhancers / promoters, splice junctions, polyadenylation signals, etc.) can be placed next to the coding region to allow correct transcription initiation and / or correct processing of primary transcripts (i.e., the coding region of the enzyme). Alternatively, the coding region used in the expression vector can contain endogenous enhancers / promoters, splice junctions, insertion sequences, polyadenylation signals, etc. or a combination of endogenous and exogenous control elements.

[0081] The expression vector generally includes a promoter, a transcription and translation initiation region, a DNA sequence encoding an enzyme, and a transcription and translation termination region that functions in the host cell in the 5'-3' direction of transcription. In one aspect, a T7-based vector can be used, which can contain at least the following components: an origin of replication, a selective antibiotic resistance gene (e.g., amp r , tetr, chlrr), multiple cloning site, T7 initiator and terminator sequences, ribosome binding site and T7 promoter.

[0082] In general, any suitable promoter that can be operably connected to heterologous DNA can be used so that the transcription of DNA can be initiated from the promoter by RNA polymerase, which can specifically recognize, bind to and transcribe the DNA in the open reading frame. Some useful promoters include constitutive promoters, inducible promoters, promoters of regulation, cell-specific promoters, viral promoters and synthetic promoters. In addition, although the promoter can include the sequence that RNA polymerase binds, this is not a requirement. Promoters can be obtained from a variety of different sources. For example, promoters can be derived completely from the natural genes of host cells, composed of different elements derived from different promoters found in nature, or composed of completely synthetic nucleic acid sequences. Promoters can be derived from many different types of organisms and customized for use in a given cell. For example, in addition to participating in the region (including the coding sequence) controlling protein translation, promoters can include the region that other regulatory proteins can bind.

[0083] The translation initiation sequence can be derived from any source, for example, any expressed E. coli gene. Typically, the gene is a highly expressed gene. The translation initiation sequence can be obtained by standard recombinant methods, synthetic techniques, purification techniques, or combinations thereof, which are well known. Alternatively, the translation initiation sequence can be obtained from many commercial suppliers. (Operon Technologies; Life Technologies Inc.).

[0084] Termination region can be natural to the transcription initiation region, can be natural to the coding region, or can derive from another source. The transcription termination sequence recognized by the transformed cell is a regulatory region that is positioned at the 3' of the translation termination codon, and is therefore positioned at the flank of the coding sequence together with the promoter. Example comprises the transcription termination sequence that derives from the gene with a strong promoter, such as the trp gene and other biosynthetic genes in Escherichia coli.

[0085] The vectors that can be used include, but are not limited to, those vectors that can be replicated in prokaryotes and eukaryotes. For example, vectors that replicate in bacteria, yeast, insect cells, and mammalian cells can be used. Examples of vectors include plasmids, phagemids, phages, viruses (e.g., baculoviruses), cosmids, and F-factors. Specific vectors can be used for specific cell types. In addition, shuttle vectors can be used for cloning and replication in more than one cell type. Such shuttle vectors are known in the art. If necessary, the vector can be a bifunctional expression vector that can function in multiple hosts.

[0086] As required, the expression vector encoding the extremophilic enzyme (such as thermophilic enzyme) can be introduced into the host cell by any method known to those skilled in the art, and the nucleic acid construct can be carried extrachromosomally in the host cell or can be integrated into the host cell chromosome. The carrier for prokaryotic host (such as bacterial cell) includes a replication system so that it can be maintained in the host for expression or for cloning and amplification. The carrier can be present in the cell with high copy number or low copy number. Usually, there are about 5 to about 200 and usually about 10 to about 150 high copy number vectors in the host cell. The host cell containing the high copy number vector preferably contains at least about 10 and more preferably at least about 20 plasmid vectors. Usually, there are about 1 to 10 and usually about 1 to 4 low copy number vectors in the host cell.

[0087] In various aspects, bacteria are used as host cells. Examples of bacteria include, but are not limited to, Gram-negative organisms and Gram-positive organisms. In one aspect, an E. coli expression system suitable for T7 protein expression can be used. Examples of T7 expression strains may include, but are not limited to, BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, Tuner(DE3)pLysS, Tuner(DE3), Lemo21(DE3), NiCO2(DE3), Oragami2(DE3), Origami B(DE3), ShuffleT7 Express, HMS174(DE3), HMS174(DE3)pLysS, DH5aplhaE, Rosetta2(DE3), Rosetta2(DE3)pLysS, NovaBlue(DE3), Rosetta-gami B, Rosetta-gami B(DE3), Rosetta-gami B(DE3)pLysS, Rosetta Blue(DE3), Novagen(DE3), Novagen(DE3)pLysS.

[0088] Expression vectors can be introduced into bacterial cells by conventional transformation / infection procedures. Nucleic acid constructs containing expression cassettes can be integrated into the genome of bacterial host cells using integration vectors. Integration vectors usually contain at least one sequence homologous to the bacterial chromosome that allows vector integration. Integration vectors can also contain phage or transposon sequences. Extrachromosomal vectors and integration vectors can contain selective markers to allow selection of transformed bacterial strains.

[0089] Useful E. coli expression system vectors can contain constitutive or inducible promoters to guide the expression of fusion or non-fusion proteins. For fusion vectors, many amino acids are usually added to the target gene sequence of expression. In addition, proteolytic cleavage sites can be introduced at the site between the target recombinant protein and the fusion sequence. Once the fusion protein has been purified, the cleavage site enables the target recombinant protein to be separated from the fusion sequence. Enzymes suitable for cutting proteolytic cleavage sites include TEV, factor Xa and thrombin. Fusion expression vectors that can be used for the present invention can include expression such as but not limited to maltose binding protein (MBP), thioredoxin (THX), chitin binding domain (CBD), six histidine tags (His-tags) (SEQ ID NO: 3), glutathione-S-transferase protein (GST), FLAG peptides, N-utilize substances (NusA) or those of small ubiquitin modifications (SUMO) fused to the target recombinase.

[0090] Methods for introducing exogenous DNA into host cells are available in the art and may include transformation of bacteria treated with CaCl or other reagents such as divalent cations and DMSO. DNA may also be introduced into host cells by electroporation, use of bacteriophage, ballistic transformation, calcium phosphate coprecipitation, protoplast fusion, electroporation, treatment of host cells with lithium acetate or by electroporation. Transformation procedures are usually different depending on the bacterial species to be transformed.

[0091] After nucleic acid is transformed or transfected to cell, the cell in which nucleic acid exists can be selected by using a selective marker. Selective markers are usually encoded on the nucleic acid introduced into the recipient cell. However, during nucleic acid is introduced into the host cell, the cotransfection of a selective marker can also be used. The selective marker that can be expressed in the recipient host cell can include but is not limited to genes that make the recipient host cell resistant to drugs (such as actinomycin C1, actinomycin D, amphotericin, ampicillin, bleomycin, carbenicillin, chloramphenicol, geneticin, gentamicin, hygromycin B, kanamycin sulfate, methotrexate, mitomycin C, neomycin sulfate, sodium novobiocin, sodium penicillin G, puromycin hydrochloride, rifampicin, streptomycin sulfate, tetracycline hydrochloride and erythromycin). Selective markers can also include biosynthetic genes, such as those genes in histidine, tryptophan and leucine biosynthetic pathways. After transfection or transformation of host cells, cells are placed in contact with appropriate selection agents.

[0092] When modifying a microorganism, any suitable Gram-positive or Gram-negative bacteria may be used. For example, the modified bacteria may be obtained from the genus Streptomyces. Specific examples of microorganisms from the genus include Streptomyces thermovulgaris, Streptomyces thermoolivaceus, Streptomyces thermohygroscopicus, Streptomyces thermocarboxydovorans, or mixtures thereof.

[0093] The following genera may also be selected to express the enzymes of the present invention according to the present disclosure:

[0094] Firmicutes: Bacillus, Xulihua, and Clostridium;

[0095] Proteobacteria: Bradyrhizobium, Sphingomonas, Azotobacter, Azospirillum, Nitrobacter, Lysobacter, Stenotrophomonas, Rhizobium, Acinetobacter, Thiobacillus, Schlegelella, Janthinobacterium, Sinorhizobium, Pseudomonas, Agrobacterium, and Escherichia (e.g., Escherichia coli);

[0096] Actinobacteria: Rhodococcus, Arthobacter, Streptomyces, Conexibacter, Rhodococcus, Solirubrobacter, Micrococcus, Rubrobacter, and Actinomyces;

[0097] Bacteroidetes: Flavobacterium and Pedobacter;

[0098] Deinococcus-thermus: Deinococcus and Thermus;

[0099] Gemmatimonads: Gemmatimonas and Gemmatironas;

[0100] Spirochaetes: Tumeriella and Leptospira;

[0101] Verrucomicrobia: Pedosphaera, Chthoniobacter and Verrucomicrobia;

[0102] Chloroflexi: Thermogemmatispora and Dictyobacter; and

[0103] Armatimonadetes: Fimbriimonas

[0104] It should be understood that the following list is merely exemplary. Specific genera may be selected based on temperature, oxygen availability, salinity, other environmental characteristics, and the like.

[0105] The following organisms can also be selected according to the present disclosure to express enzymes (e.g., purified enzymes) of the present disclosure: Lysobacter estuarineus, Lysobacter antibioticus, Lysobacter bugur, Lysobacter capsici, Lysobacter enzyme-producing, Lysobacter lacus, Lysobacter lycopersici, Lysobacter mali, Lysobacter niastensis, Lysobacter deep-source, Lysobacter species, Lysobacter species A03, Lysobacter species cf310, Lysobacter species H21R20, Lysobacter species H21R4, Lysobacter species H23M41, Lysobacter species R19, Lysobacter species Root604, Lysobacter species Root690, Lysobacter species Root916, Lysobacter species Root983, Lysobacter species TY2-98, Lysobacter sponge, Lysobacter sponge, Lysobacter, Lysobacter alkaline soil, Lysobacter resistant to arsenic, Lysobacter datiana, Lysobacter dokdonensis, Lysobacter zymogenes, Lysobacter zymogenes, Lysobacter gilvus, Lysobacter colloidus, Lysobacter mali, Lysobacter oculi, Lysobacter panacisoli, Lysobacter penaei, Lysobacter prati, Lysobacter psychrotolerant, Lysobacter pythonis, Lysobacter swiss, Lysobacter segetis, Lysobacter silvestris, Lysobacter silvisoli, Lysobacter soli, Lysobacter species, Lysobacter species 17J7-1, Lysobacter species Alg18-2.2, Lysobacter species Cm-3-T8, Lysobacter species H23M47, Lysobacter species HDW10, Lysobacter species II4, Lysobacter species N42, Lysobacter species OAE881, Lysobacter species Root494, Lysobacter species URHA0019, Lysobacter species WF-2, Lysobacter species yr284, Lysobacter tabacisoli, Lysobactertelluris, Lysobacter tolerans, Lysobacter tolerans, Lysobacter xinjiang, Unclassified Lysobacter, Aliivibrio finisterrensis, Aliivibrio finisterrensis, sifiae, Allovibrio species, Allovibrio species 1S128, Allovibrio species EL58, Allovibrio species SR45-2, Caballeronia arvi, Caballeroniacalidae, Caballeronia hypogeia, Caballeroniainsecticola, Caballeronia pedi, Caballeronia terrestris, Dokdonella koreensis, Dyella caseinilytica, Dyellachoica, Dyella flava, Dyella kyungheensis, Dyellamobilis, Dyella monticola, Dyella nitratireducens, Dyella psychrodurans, Dyellasoli, Dyella solisilvae, Dyella species 7MK23, Dyella species ASV21, Dyella species ASV24, Dyella species C11, Dyella species C9, Dyella species DHC06, Dyella species EPa41, Dyella species G9, Dyella species M7H15-1, Dyella species M7H15-1, Dyella species OK004, Dyella species S184, Dyella species SG562, Dyella species SG609, Dyella species YR388, Dyella tabacisoli, Fluorescent Bacterium bozemanense, Fluorescent Bacterium duomogenes NY 23. Fluorescent Bacillus gordonii, Microoscillatoria maritima, Pseudomonas aeruginosa, Pseudomonas thermotolerant, Pseudomonas mediterranea, Psychrobacter species, Psychrobacter species MB-3u-54, Psychrobacter species psych-6C06, Psychrobacter species RZ22, Psychrobacter species Urea-02u-13, Denitrifying Rhodobacter, Rhodobacter fuchsii, Rhodobacter licoriceus, Rhodanobacter lindaniclasticus, Rhodanobacter panaciterrae, Rhodanobacter species 7MK24, Rhodanobacter species A1T4, Rhodanobacter species B04, Rhodanobacter species B05, Rhodanobacter species C01, Rhodanobacter species C03, Rhodanobacter species C05, Rhodanobacter species C06, Rhodanobacter species DHB23, Rhodanobacter species DHG33, Rhodanobacter species L36, Rhodanobacter species MP1X3, Rhodanobacter species OK091, Rhodanobacter species OR444, Rhodanobacter species PCA2, Rhodanobacter species Root480, Rhodanobacter species Root627, Rhodanobacter species Root627, Rhodanobacter species SCN67-45, Rhodanobacter species SCN 68-63, Soil772, T12-5, TND4EH1, TND4FH1, Rhodanobacterspathiphylli, Rhodanobacter thiooxydans, Stenotrophomonas chelatiphaga, Oligotrophomonas maltophilia, Stenotrophomonas panacihumi, Stenotrophomonas pavanii, Rhizotrophomonas rhizophila, Stenotrophomonas species DDT-1, Stenotrophomonas species RIT309, Stenotrophomonas species SKA14, Estuarine Vibrio, Vibrioantiquaries, Aquatic Vibrio, Tasmanian Vibrio, Xanthomonasales, Albiflora Xanthomonas, Tree Xanthomonas, Carpetgrass Xanthomonas, Bromo Xanthomonas, Campesino Xanthomonas, Cannabis Xanthomonas, Citrus Xanthomonas, Eufoam Xanthomonas, Strawberry Xanthomonas, Geranium Xanthomonas, Hyacinthus Xanthomonas, Rice Xanthomonas, Bean Xanthomonas, Pea Xanthomonas, Sugar Xanthomonas, Xanthomonas species Leaf131, Xanthomonas species (NCPPB 1128), Xanthomonas translucens, Xanthomonas vasicola, Xanthomonasvesicatoria, or a combination thereof. It should be understood that the following list is exemplary only. Specific microorganisms may be selected based on temperature, oxygen availability, salinity, other environmental characteristics, and the like.

[0106] In another aspect, the methods disclosed herein can include enzymes that effectively work together in the same environment, which is characterized by the same or similar pH and temperature. For example, more than one enzyme can be selected to work well in a temperature range of, for example, about 60°C to about 100°C and a pH range of about 5-7, or any suitable temperature and pH combination thereof.

[0107] In some aspects, fungal enzymes can be used for the methods of the present disclosure. For example, fungal long-chain alcohol dehydrogenases can be used, which the inventors have found to greatly accelerate the reaction, although not in thermophilic processes. However, such non-thermophilic enzymes can be added to the reactions of the present disclosure after the temperature has been lowered.

[0108] Other fungal sources of enzymes that may be utilized in the present disclosure include, but are not limited to, Aureobasidium, Macroventuria, Lophium, Tothia, Trichodelitschia, Westerdykella, Didymosphaeria, Viridothelium, Delitschia, Zopfia, Myriangium, Rhizodiscina, Saccharata, Aaosphaeria, Amniculicola, Byssothecium, Aspergillus, lus, Meira, Dissoconium, Lizonia, Aureobasidium, Morchella, Sodiomyces, Tilletiopsis, Jaminaea, Ceraceosorus, Testicularia, Tilletiopsis, Violaceomyces, Rhizopus, Alternaria, Hesseltinella, Neurospora, Ramularia, and Rhynchosporium.

[0109] Regardless of the enzyme selected, as discussed above, the bioplastic polymer produced in the disclosed method is a polyalkanoate. For example, the polyalkanoate is a polyhydroxyalkanoate or a polyhydroxybutyrate. The polyhydroxyalkanoate produced can be characterized as being able to react chemically in a manner comparable to that of polypropylene or polyethylene. The polyhydroxyalkanoate can include any one or more of the following carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30.

[0110] In one aspect, the PHA monomers or polymers can have linear carbon chains. In another aspect, the PHA monomers and / or polymers can be at least about 80% homogeneous, such as at least about 85% homogeneous, at least about 90% homogeneous, at least about 95% homogeneous, or at least about 99% homogeneous. However, in one aspect, it is understood that the chain length of the PHA monomers and / or polymers produced is directly related to the chain length of the long-chain fatty acids or their precursors.

[0111] In another aspect, the mass yield of PHA monomers and / or polymers can be directly proportional to the optical density measurement obtained. For example, the higher the optical density reading, the higher the mass yield of the PHA monomers and / or polymers produced. In another aspect, PHA monomers and / or polymers can contain substantially the lowest amount of any microplastics and / or nanoplastics. For example, containing substantially the lowest amount of any microplastics and / or nanoplastics can include an amount of about 0.01% to about 10% of the total mass yield, such as about 0.1% to about 9% of the total mass yield, about 1% to about 8% of the total mass yield, about 2% to about 7% of the total mass yield, about 3% to about 6% of the total mass yield, or about 4% to about 5% of the total mass yield of microplastics and / or nanoplastics. In other aspects, the bioplastic polymer produced in step d) can be substantially free of any microplastics and / or nanoplastics. For example, if present, any microplastics and / or nanoplastics can be present in the produced PHA monomers and / or polymers in an undetectable amount.

[0112] In certain aspects, the PHA monomer and / or polymer can be a polyhydroxyalkanoate homopolymer, which includes poly 3-hydroxyalkanoate (e.g., poly 3-hydroxypropionate (PHP), poly 3-hydroxybutyrate (PHB), poly 3-hydroxyvalerate (PHV), poly 3-hydroxyhexanoate (PHH), poly 3-hydroxyoctanoate (PHO), poly 3-hydroxydecanoate (PHD) and poly 3-hydroxy-5-phenylvalerate (PHPV)), poly 4-hydroxyalkanoate (e.g., poly 4-hydroxybutyrate (PHB) and poly 4-hydroxyvalerate (hereinafter referred to as PHB)) or poly 5-hydroxyalkanoate (e.g., poly 5-hydroxyvalerate (hereinafter referred to as PHV)).

[0113] In certain aspects, PHA can be a copolymer (containing two or more different monomer units), wherein the different monomers are randomly distributed in the polymer chain. Examples of PHA copolymers can include poly 3-hydroxybutyrate-co-3-hydroxypropionate (hereinafter referred to as PHB3HP), poly 3-hydroxybutyrate-co-4-hydroxybutyrate (hereinafter referred to as P3HB4HB), poly 3-hydroxybutyrate-co-4-hydroxyvalerate (hereinafter referred to as PHB4HV), poly 3-hydroxybutyrate-co-3-hydroxyvalerate (hereinafter referred to as PHB3HV), poly 3-hydroxybutyrate-co-3-hydroxyhexanoate (hereinafter referred to as PHB3HH), and poly 3-hydroxybutyrate-co-5-hydroxyvalerate (hereinafter referred to as PHB5HV), etc., with a central carbon chain length of up to C30 as discussed above.

[0114] In other aspects, the present disclosure relates to an abiotic method for producing PHA monomers and / or polymers from long-chain fatty acids or their precursors. For example, long-chain fatty acids or their precursors can be contacted in vitro with purified enzymes or purified enzyme mixtures to produce PHA monomers and / or polymers. For example, the enzymatic reaction of the abiotic method can be carried out in a single container or in more than one container. In addition, the abiotic method can be carried out at a temperature range of about 40°C to about 80°C, or any variant thereof as described above.

[0115] In one aspect, the abiotic method can include one or more additional steps: for each of the one or more additional steps, long-chain fatty acids or its precursor are contacted with enzymes or enzyme mixtures in vitro by repeating the enzymatic step. For example, one or more additional steps can be repeated at least three times. The abiotic method can include contacting long-chain fatty acids or its precursor with a first enzyme in vitro, then contacting one or more subsequent enzymes in a stepwise manner, or the abiotic method can include contacting long-chain fatty acids or its precursor with two or more enzymes in vitro simultaneously. For example, one or more subsequent enzymes can include at least one additional enzyme different from the first enzyme. Of course, as mentioned above, in one aspect, it should be understood that the present disclosure also includes a kind of abiotic method that long-chain fatty acids or its precursor are contacted with all necessary enzymes simultaneously. However, in such an aspect, if one or more enzymes added simultaneously need to be refreshed, one or more enzymes can be repeatedly added.

[0116] In another aspect, the PHA monomers and / or polymers produced in the abiotic method can include polyalkanoates. For example, the polyalkanoates can be polyhydroxyalkanoates and / or polyhydroxybutyrates. The polyhydroxyalkanoates can be characterized as chemically reacting in a manner comparable to that of polypropylene or polyethylene. The polyhydroxyalkanoates can have any one or more of the following carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30.

[0117] In one aspect, the PHA monomers and / or polymers produced in the organism-free process can include linear carbon chains. In one aspect, the PHA monomers and / or polymers produced in the organism-free process can be at least 80% homogeneous, or any variation thereof as discussed above. In another aspect, the mass yield of the PHA monomers and / or polymers produced in the organism-free process can be directly proportional to the optical density measurement obtained. In another aspect, the PHA monomers and / or polymers produced in the organism-free process can contain substantially minimal amounts of any microplastics and / or nanoplastics.

[0118] In one aspect, the enzyme or enzyme mixture in the organism-free method can be purified from extreme microorganisms, as described herein. The microorganism that can be purified from enzyme can be an engineered microorganism, which has been genetically modified to secrete the specific enzyme for the organism-free method, or the microorganism that can be purified from enzyme can be at least one type of naturally occurring microorganism, the naturally occurring microorganism natural encoding specific enzyme. For example, enzyme or enzyme mixture can be a thermophilic enzyme. The thermophilic enzyme can tolerate the temperature of about 40 ℃ to about 120 ℃ or any variant thereof as discussed above. For example, at least one enzyme can be purified from thermophilic Xu Lihua bacteria. In one aspect, the enzyme mixture used can include an enzyme that effectively synergizes in the same environment, and the same environment is characterized by identical or similar pH and temperature, as described above.

[0119] In another aspect, the organism-free method can include contacting one or more long-chain fatty acids or their precursors with a purified enzyme or purified enzyme mixture in an environment substantially free of any bacteria that secrete the same enzyme or the same enzyme mixture to produce linear PHA monomers and / or polymers. For example, the linear PHA monomers and / or polymers can include polyhydroxyalkanoates having a carbon chain length greater than C8 and / or a carbon chain length less than C30.

[0120] In yet another aspect, generally, the present disclosure relates to uncharacterized polyhydroxyalkanoates. For example, the carbon chain length of the uncharacterized polyhydroxyalkanoate can be greater than C8, and can have a straight-chain polymer substantially free of any side chain pendant polymers. For example, the carbon chain length of the uncharacterized polyhydroxyalkanoate can be greater than C8 but less than C30, and can have a straight-chain polymer.

[0121] In another aspect, the disclosure generally relates to a method for producing polyalkanoates in a multi-step enzymatic reaction. For example, the method may include contacting a long-chain fatty acid (if it is a precursor thereof, contacting with an essential enzyme to obtain a long-chain fatty acid), contacting a long-chain fatty acid with a long-chain fatty acid CoA ligase / synthetase to obtain a long-chain fatty acid acyl-CoA, contacting a long-chain fatty acid acyl-CoA with a long-chain acyl-CoA dehydrogenase, then contacting with a long-chain-enoyl-CoA hydratase, then contacting with a hydroxyacyl-CoA dehydrogenase to obtain a long-chain acetoacetyl-CoA, and contacting a long-chain acetoacetyl-CoA with a hydroxyacyl-CoA dehydrogenase to obtain a hydroxyacyl-CoA for polymerization into polyhydroxyalkanoates, or contacting a long-chain acetoacetyl-CoA with an acetyl-CoA C-acyltransferase to obtain acetyl-CoA, and contacting acetyl-CoA with an acetoacetyl-CoA synthetase to obtain a polyhydroxyalkanoate (in this example, such as PHB). Of course, as described above, in one aspect, each of the enzymes mentioned above can be added simultaneously. That is, as discussed, by careful selection of compatible enzymes, such as those discussed above, a one-pot reaction is possible, allowing all enzymes to be added simultaneously, such that the reaction proceeds naturally from long-chain fatty acids or their precursors to produce PHAs without further intervention. However, as discussed, in one aspect, one or more of the simultaneously added enzymes can be added or "refreshed" during the one-pot process.

[0122] In some aspects, the long-chain fatty acid CoA ligase / synthetase is purified from the halophilic thermosporium or any other microorganism described herein. For example, the alcohol dehydrogenase can be a fungal long-chain alcohol purified dehydrogenase as described herein.

[0123] The present disclosure may be better understood with reference to the following examples.

[0124] Example

[0125] The present disclosure may be better understood by reference to the following examples.

[0126] Materials and Methods

[0127] source : Enzyme sequences were identified by searching the NCBI protein or equivalent proteome databases. For this example, it was decided to focus primarily on the thermophilic and halophilic Thermospora halophila. The sequences and accession numbers of the enzymes used in this work are listed in Appendix I. All general laboratory chemicals and long-chain fatty acids were purchased from Millipore-Sigma Chemical Company (St. Louis, MO). Cloning materials and protein purification supplies were from New England Biolabs (Beverly, MA).

[0128] Enzyme cloning : The amino acid sequence of each enzyme was searched for potential signal sequences. If a signal sequence was present, it was removed from the sequence. A histidine tag and TEV protease cleavage sequence were added to the N-terminus of each enzyme. The sequence was MHHHHHHENLYFQS to aid purification (via an NTA-Ni column) and subsequent removal of the hexahistidine tag. Since TEV protease cuts after glutamine (Q), each recombinant enzyme starts from an N-terminal serine residue. Each amino acid sequence was reverse translated into an E. coli codon-optimized nucleic acid sequence and synthesized using standard laboratory gene synthesis techniques. The insert sequence was digested with Nde I and cloned into a similarly digested expression vector pET11d. The final DNA sequence was verified by DNA sequencing. The expression plasmids were used to transform E. coli Origami2 (DE3) for expression

[0129] Protein expression and purification : Use a single colony to inoculate 5.0 mL of LB amp Culture medium (80 mg / mL ampicillin concentration). Grow the bacteria at 37°C for 12 hours and use this culture to inoculate 500 mL of LB ampCulture medium. The culture is grown at 37°C until the optical density (600nm) reaches 0.4 (usually in 3-5 hours). Protein expression is induced by adding IPTG to a final concentration of 1mM. The cells are grown for 12 hours at 37°C. The cells are harvested by centrifugation at 5,000xg for 20 minutes and frozen at -80°C until use (the shortest freezing time is 24 hours). The cells are thawed on ice and resuspended in buffer A (0.5M NaCl, 20mM Tris-HCl, 5mM imidazole, pH 7.9) (usually 1mL per gram of cells). The cells are crushed by passing through a French crusher twice, and then centrifuged at 30,000xg for 30 minutes. The crude extract is mixed with an equal volume of charged His-Bind resin slurry, and the mixture is poured into a 5cm x 4.9cc chromatographic column. The chromatographic column was washed with 10 column volumes of wash buffer (0.5M NaCl, 20mM Tris-HCl, 60mM imidazole, pH 7.9) at a flow rate of 0.2mL / min. The enzyme was eluted from the chromatographic column by adding 3 column volumes of 0.5M NaCl, 20mM Tris-HCl, 1.0M imidazole, pH 7.9. Fractions (1.0mL) were collected. The fractions containing the enzyme were merged after being analyzed by SDS PAGE. The merged fractions were applied to a 70cm x 4.9cc Sephadex G-75 column (10mM Tris-HCl, pH 7.5, 1mM EDTA). The fractions containing homogeneous protein were merged (after being checked by SDS PAGE) and concentrated to 5mg / mL by a Centricon filter. The enzyme was stored frozen at -20°C until use. The TRX-histidine tag region was removed from the enzyme using TEV protease. The protein was diluted to 10 mM Tris-HCl pH 7.5, 25 mM NaCl at 1.0 mg / mL. 100 U TEV protease was added per mg of enzyme (ratio of approximately 1:100 (w / w)). The reaction was allowed to proceed at 4°C for 16 hours. The mixture was passed through a charged nickel chromatography column. One column volume of the eluate was collected to represent the purified tag-free enzyme.

[0130] Enzyme assay:The formation of long-chain fatty acid CoA was monitored by measuring the amount of pyrophosphate released in the reaction using the MAK168 pyrophosphate assay kit from Millipore-Sigma Chemical Co (St. Louis, MO). The formation of PHA was monitored by the increase in optical density (650 nm) over time in a Molecular Devices SpectraMax 5 spectrometer. The assay measures the increase in light scattering due to precipitated PHA formed during the reaction. The depolymerization of the isolated PHA material was performed using a previously characterized thermophilic Xulihua PHB depolymerase (Quirk, 2020.TL-23500). Standard reaction conditions were 10 mM KCl, 10 mM MgCl2, 20 mM CHES (pH 9.0), 55 ° C.

[0131] Turbidimetric assay was used to measure PHBD enzyme activity under various conditions. Standard reactions (final volume = 1.0 mL) contained 200 mg / L PHB particles (previously stabilized by sonication), 1 mM CaCl2, 10 mM KCl, 0.5 M NaCl, 20 mM buffers at different pH values. Reactions were started after addition of the enzyme and monitored at 650 nm in absorbance mode in an Applied Photophysics spectropolarimeter. The reactions were gently stirred and maintained at a constant temperature. OD measurements (usually starting in the range of 2-3) were converted to percentages of residual OD over time. All reported kinetic constants are according to Segel (1993).

[0132] The reduction of fatty acid amount in the initial reaction was measured using a fatty acid quantitative kit from Millipore-Sigma Chemical Co. During the initial reaction, aliquots were taken out at time and the remaining fatty acid content was measured using the kit. The fluorescence emission intensity at 587 nm (excitation wavelength 535 nm) was measured on a Molecular Devices SpectraMax 5 spectrometer.

[0133] Example 1

[0134] Using the enzymatic fatty acid assay, the long-chain fatty acid-CoA ligase reaction of two different fatty acids was measured ( Figure 2 The time course of these reactions is Figure 6The figure shows the conversion of arachidic acid (C18) and myristic acid (C12). Although the kinetics of the reactions differ slightly during the assay, both reactions were completed within two hours under the conditions tested. The formation of pyrophosphate released in the formation of long chain fatty acid CoA molecules occurs simultaneously with the measurable reduction of fatty acids in the reaction. The time course of the reaction fully reflects the reduction of fatty acids. This is shown in Figure 7 Thus, the key reactions in the present disclosure have been clearly demonstrated for two medium-length fatty acids.

[0135] Once the long-chain fatty acid CoA portion is produced, it is used as Figure 4 Inputs to the reactions shown. The overall 5-step reaction is easily monitored by the formation of PHA precipitating from solution and alleviates the need to track the progress of any or all other enzymatic steps. Figure 8 A measurable increase in optical density due to light scattering (650 nm) is shown after a lag time of approximately 50 minutes. This lag time corresponds to the time required to complete the first four enzymatic steps and accumulate enough substrate for the final reaction. The formation of precipitated PHA is then linear over the next 150 minutes before the reaction ends for arachidic acid (as starting material) or slows down biphasically when myristic acid is used as the starting substrate. Although the slopes of the linear portion of the reaction are almost identical, it is unclear what drives the behavior at the end of the assay. It may be that substrate depletion begins to affect (measured in k cat / K m The main observation is that the precipitation material can be efficiently produced using this scheme.

[0136] The precipitated PHA material was separated from the reaction components by brief centrifugation and two rounds of phosphate-buffered saline washing. The final precipitated material was resuspended in depolymerase reaction buffer (see Methods) and mixed with PHB depolymerase from X. thermophila. Fig. 9 As shown, PHA materials formed using either arachidic acid or myristic acid can depolymerize over the course of an hour, as measured by a decrease in light scattering. Myristic acid-based reactions can be driven to near completion (as evidenced by light density approaching zero at 60 minutes), while arachidic acid-based reactions end with some undegraded material remaining.

[0137] Example 2

[0138] according to Figure 2 and Figure 3 The overall enzymatic steps shown form homogeneous chain-length PHAs from two single chain-length fatty acids. That is, equimolar amounts of arachidic acid and myristic acid are completely converted to their respective CoA moieties using the WP_068688876 enzyme. This is followed by the formation of pyrophosphate, Fig.10 The time course indicated that the reaction was complete within 90 minutes. The two CoA partial pools were then used as substrates for the formation of mixed C12 / C18 PHA polymers, as indicated by an increase in light scattering (as material precipitated from the overall reaction over time) ( Fig.11 After the overall reaction is complete, the separated mixed C12 / C18 PHA polymers are subjected to depolymerization using a thermophilic Xulihua PHA depolymerase, such as Fig.12 As shown. The reaction is characterized by a short lag phase (approximately 10 minutes) followed by intense depolymerization and a gradual slowing of the reaction rate after 55 minutes. After one hour, approximately 12% of the input mixed polymer remained. This most likely reflects the substrate preference of the single depolymerase used in this reaction. Nevertheless, this does confirm that polymers are formed in the mixed substrate situation that can be depolymerized by known PHA depolymerases. As shown, substrates can be mixed and matched to produce new PHAs that are multiples of the input. In this case C12 / C18. By varying the substrate input ratios, new PHAs with compositions reflecting these ratios can be produced.

[0139] Example 3

[0140] Arachidic acid and myristic acid represent roughly the middle range of available fatty acids (see e.g., Figure 3 To test whether a wider range of chain lengths could be exploited, the C24 fatty acid hexacosanoic acid was used for PHA formation. Formation of the CoA moiety using the WP_068689753 enzyme was significantly slower than the other reactions ( Fig.13 ); characterized by a 50-minute lag time and completed after 240 minutes. However, the reaction still proceeded and the coenzyme A formed was partially converted into a measurable precipitated polymer, such as Fig.14 shown.

[0141] Interestingly, the hexacosanoic acid-derived polymers exhibited different depolymerization when two different PHA depolymerases were used in the depolymerization reaction. The depolymerase from Thermus thermophilus was significantly more capable of depolymerizing the polymer than the enzyme from P. thermophilus. Fig.15 As shown. The polymer can be degraded by both enzymes, but at different rates and extents. Both enzymes leave unreacted polymer and / or insoluble reaction byproducts. Therefore, it should be noted that just as with the selection of enzymes for the formation of long chain fatty acid CoA molecules, it is also important to use an ideal / compatible depolymerase as part of further polymer studies.

[0142] Example 4

[0143] Shorter chain length fatty acids were also used to generate new PHAs. Heptanoic acid (C5) was readily converted to the CoA moiety when the WP_068692787 enzyme was used. Fig.16 , that is, it illustrates the formation of pyrophosphate in the initial reaction of heptanoic acid.

[0144] The optical density of the substrates PHA and heptanoic acid-CoA was measured using Fig.17 ), indicating the formation of PHA, which precipitates from solution over time. Fig.17 Similarly, PHA was rapidly depolymerized using the thermophilic Xulihua PHB depolymerase, as shown in Fig.18 shown.

[0145] Example 5

[0146] New homogeneous PHA polymers and copolymers were formed using various enzymes isolated from Halomonas titanicus and three fatty acid starting substrates with different chain lengths: octanoic acid (C6), myristic acid (C12), and behenic acid (C20). Enzymes were added to a final concentration of 20 μg / mL and fatty acid starting substrates were added to a final concentration of 10 mg / mL. The reaction buffer contained 10 mM Tris-HCl (pH 7.5), 5 mM ATP, 10 mM MgCl2, 10 mM KCl, 10 mM MnCl2, 10 mM Coenzyme A, 10 mM flavin adenine dinucleotide, 1.0 M NaCl, 5 μg / mL Titanicus inorganic pyrophosphatase (EC 3.6.1.1), 10 mM NADH, 20 μg / mL polyphosphate:AMP phosphotransferase (EC 2.7.4.3x, Halomonas halophila), 10 mg / mL linear polyphosphate, isocitrate dehydrogenase (EC 1.1.1.41 / 42, Titanicus) and 100 mM isocitrate.

[0147] First, the long-chain fatty acids were converted into long-chain fatty acid CoA molecules ( Figure 2 The reaction shown). Halomonas titanica expresses ten fatty acid-CoA ligases, several of which are nearly identical. The percent identity matrix of the remaining five most unique fatty acid-CoA ligases (QKS24074, QKS24113, QKS25406, QKS26753, and QKS23364) expressed by Halomonas titanica indicates that these five most unique ligases are 22% to 32% identical (Table I). QKS24074 and QKS24113 are the least identical enzymes, 22.18% identical, while QKS26753 and QKS23364 are the most similar enzymes, 32.99% identical. Fig.19 is a consensus maximum likelihood tree further illustrating the relationships among the five most unique fatty acid-CoA ligases expressed by Halomonas titanicola. The plethora of available enzymes suggests that care must be taken in selecting the appropriate enzyme to match the chain length of long-chain fatty acids.

[0148] Table I. Percent identity matrix - fatty acid CoA ligases.

[0149]

[0150] Although enzymes can be selected to run Figure 2 The reactions shown were performed, but the best enzyme was selected for each of the three chain length fatty acids from the following five Titanicus Halomonas fatty acid-CoA ligases: QKS24074, QKS24113, QKS25406, QKS26753, and QKS23364. The substrate chain length preference distribution (activity percentage relative to carbon chain length) for each of the five fatty acid-CoA ligases is shown in Table II. For octanoic acid (C6): The results show that QKS24074 is the best enzyme; QKS23364 is a possible enzyme, but with much lower activity; and QKS25406, QKS24113, and QKS26753 are unlikely enzymes. For myristic acid (C12): the results show that QKS26753 or QKS23364 are the best enzymes, while QKS25406, QKS24113 or QKS24074 are possible but with much lower activity. For behenic acid (C20): the results show that QKS25406 or QKS24113 are the best enzymes, QKS23364 is a possible enzyme but with much lower activity, while QKS24074 and QKS26753 are unlikely enzymes. The inventors have found that while the reaction can be run with enzymes that are much less active towards fatty acids, this is far from preferred. The inventors have also found that the reaction can be run with one or more than one fatty acid CoA ligase in the presence of two moieties with different chain lengths.

[0151] Chain length QKS25406 QKS24113 QKS24074 QKS26753 QKS23364 C6 0.0 0.0 100 0.0 25.0 C8 0.0 0.0 100 0.0 70.7 C10 0.0 0.0 83.3 66.6 100 C12 11.6 5.2 21.0 100 100 C14 30.4 21.1 0.0 100 100 C16 100 97.5 0.0 42.4 100 C18 100 100 0.0 5.6 100 C20 100 100 0.0 0.0 46.5 C22 89.9 100 0.0 0.0 20.2

[0152] The long-chain fatty acid-CoA ligase reaction of three different fatty acids was measured using an enzymatic fatty acid assay ( Figure 2 The time course of these reactions is Fig. 20 Shown in. Fig. 20 Figures showing the conversion of octanoic acid (C6), myristic acid (C12), and behenic acid (C20). Although the kinetics of the reactions varied slightly over the course of the assay, all reactions were completed within two hours under the conditions tested. The formation of pyrophosphate released in the formation of long chain fatty acid CoA molecules occurs simultaneously with the reduction of measurable fatty acids in the reaction. Fig.21 The figure shows the formation of pyrophosphate in the initial reaction. Fig. 20 and 21 As shown, the time course of the pyrophosphate formation reaction completely reflects the reduction of fatty acids. Thus, the key reaction in the present disclosure has been clearly demonstrated to cover the three fatty acids of the available chain lengths.

[0153] Second, once the long-chain fatty acids CoA are produced, they are transported through Figure 3 The five-step reaction shown is converted to PHA. The overall five-step reaction is easily monitored by the formation of PHA precipitated from solution, which is Fig. 22 As shown in the figure. Fig. 22 It is shown that after a lag time of about 10 minutes, there is a measurable increase in optical density due to light scattering (650 nm), depending on which chain length fatty acid is used as the initial substrate. The lag time corresponds to the time required to complete the first four enzymatic steps and accumulate enough substrate for the final reaction. It should be noted that the lag phase in this example is shorter than a similar reaction performed under a thermophilic reaction. A shorter lag phase is a hallmark of a halophilic reaction, where the enzyme typically has a higher k cat / K m value.

[0154] The formation of precipitated PHAs then became linear over the next 100 min before the reaction ended when octanoic and behenic acids were used as starting materials or continued to increase linearly when myristic acid was used as the starting substrate. Although the slopes of the linear portion of the reaction were nearly identical, it is unclear what drives the behavior at the end of the assay, especially since there was no correlation between the overall kinetics of the reaction and the fatty acid chain length. The main observation is that precipitated material can be generated with high efficiency using this protocol.

[0155] The PHA material formed using caprylic acid, myristic acid or behenic acid is then depolymerized. Fig.23 The depolymerization of the formed PHA is shown graphically, as measured by the decrease in optical density at 650 nm (due to scattering). The myristic acid-based reaction can be driven to near completion, as evidenced by the optical density approaching zero at 60 minutes; however, the arachidic acid-based reaction ends with some undegraded material remaining. A possible explanation for the differences between these reactions could be differences in the kinetics and substrate preferences of the enzymes for the three starting materials.

[0156] The sequences of various enzymes used in the Examples are provided in Figures 5A to 5D.

[0157] This illustrates a multi-step enzymatic method for producing a variety of PHA bioplastic polymers from long-chain fatty acids or their precursors. Thus, this example illustrates the enzymatic conversion of long-chain fatty acids or their precursors into PHA monomers suitable for producing bioplastic polymers.

[0158] These and other modifications and variations of the present invention may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the present invention as more particularly described in the appended claims. In addition, it should be understood that the minor aspects of each aspect may be interchangeable in whole or in part. In addition, it will be appreciated by those of ordinary skill in the art that the above description is by way of example only and is not intended to limit the present invention as further described in such appended claims.

Claims

1. An enzymatic method for producing homogeneous chain length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbon atoms, the method comprising: contacting the long-chain fatty acid or its precursor with an enzyme or enzyme mixture in vitro; as well as Homogeneous chain length polyhydroxyalkanoate monomers and / or polymers are produced having a chain length of at least eight carbons.

2. The method of claim 1, wherein the carbon chain length of the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers is equal to the central carbon chain length of the long chain fatty acid or its precursor.

3. The method of claim 1 or 2, wherein the central carbon chain length of the long-chain fatty acid is any one of the following carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30, and / or wherein the central carbon chain length of the long-chain fatty acid precursor is configured to produce a long-chain fatty acid having any one of the following central carbon chain lengths: C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29 and C30.

4. The method of claim 1 or 2, wherein the method is performed in one vessel, or wherein the method is performed in more than one vessel.

5. The method of any one of the preceding claims, wherein the long chain fatty acid precursor is a long chain alkane, a long chain alcohol or a long chain aldehyde.

6. The method of any one of the preceding claims, wherein the enzyme or enzyme mixture contains at least one enzyme selected based on the central chain length of the long-chain fatty acids.

7. A method as described in any one or more of the preceding claims, further comprising a second long-chain fatty acid or a precursor thereof, the chain length of which is different from that of the long-chain fatty acid or a precursor thereof, wherein the homogeneous chain length polyhydroxyalkanoate comprises a copolymer having segments of the long-chain fatty acid or a precursor thereof and the second long-chain fatty acid or a precursor thereof.

8. The method according to any one of the preceding claims, wherein the method further comprises one or more further steps of contacting the long-chain fatty acid or precursor thereof with the enzyme or enzyme mixture in vitro.

9. A method as claimed in any one of the preceding claims, wherein the method comprises contacting the long chain fatty acid or a precursor thereof in vitro with a first enzyme and then with one or more subsequent enzymes in a stepwise manner.

10. The method of any one of the preceding claims, wherein the method comprises contacting the long-chain fatty acid or precursor thereof with two or more enzymes simultaneously in vitro.

11. The method of any one of the preceding claims, wherein the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers comprise any one or more of the following carbon chain lengths: C8, C9, C10, C11, C12, C13, C14, C15, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, and C30.

12. The method of any one of the preceding claims, wherein the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers have linear carbon chains.

13. The method of any of the preceding claims, wherein the homogeneous chain length polyhydroxyalkanoate monomers and / or polymers are at least about 90% homogeneous.

14. The method of any one of the preceding claims, wherein the enzyme or enzyme mixture is purified from an extremophilic microorganism, preferably wherein the microorganism is a bacterium of the genus Halomonas, Xulihuatella, Lysobacter, Alteromonas, Arthrobacter, Azospirillum, Steadybacter, Desulfovibrio, Halobacillus, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Halobacterium, Methyloligella, Micromonospora, Halobacter, Nocardia, Paracoccus, Roseochromia, Saccharomonas, Shewanella, Acidobacillus, Alkalianaerobe, Halobacteraceae, Mycelidomonas, Amycolatopsis, Georgesella, Thermoacidobacter, Thermosporium or a combination thereof.

15. The method of claim 14, wherein the microorganism is an engineered microorganism that has been genetically modified to secrete a specific enzyme.

16. The method of claim 14, wherein the microorganism is at least one type of naturally occurring microorganism that naturally encodes a particular enzyme.

17. The method of any one of the preceding claims, wherein the enzyme or enzyme mixture is a thermophilic enzyme.

18. The method of claim 17, wherein the thermophilic enzyme is resistant to temperatures of about 40°C to about 120°C.

19. The method of any one of the preceding claims, wherein the method is carried out at a temperature ranging from about 40°C to about 80°C.

20. The method of any one of the preceding claims, wherein the method comprises enzymes that effectively act synergistically in the same environment, the same environment being characterized by the same or similar pH and temperature.

21. The method of any one of the preceding claims, wherein the method is an organism-free method, wherein the long chain fatty alcohol or a precursor thereof is contacted with the enzyme or enzyme mixture in an environment substantially absent of any bacteria secreting the same enzyme or the same enzyme mixture.

22. An uncharacterized polyhydroxyalkanoate comprising: Polyhydroxyalkanoate having a carbon chain length greater than C8, wherein the polyhydroxyalkanoate is a linear polymer substantially devoid of any side chain pendant polymers.

23. A method of producing homogeneous chain length polyhydroxyalkanoate monomers and / or polymers having a chain length of at least eight carbons, the method comprising: contacting a long-chain fatty acid with a long-chain fatty acid CoA ligase / synthetase to obtain a long-chain fatty acid acyl-CoA; contacting the long-chain fatty acid acyl-CoA with a long-chain acyl-CoA dehydrogenase, then with a long-chain-enoyl-CoA hydratase, and then with a hydroxyacyl-CoA dehydrogenase or a long-chain-3-hydroxyacyl-CoA dehydrogenase to obtain a long-chain acetoacetyl-CoA; and The long chain acetoacetyl-CoA is contacted with acetoacetyl-CoA reductase to obtain hydroxyacyl-CoA for polymerization into polyhydroxyalkanoate.

24. The method of claim 23, wherein the long-chain fatty acid CoA ligase / synthetase is purified from the halophilic Thermospora fissifolia.

25. The method of claim 23, wherein the alcohol dehydrogenase is a fungal long-chain alcohol dehydrogenase purified from the group of fungal genera including: Aureobasidium, Venturia, Lophium, Tothia, Trichodelitschia, Westerdykella, Sphaerotheca, Viridothelium, Cryptocoryne, Zopfia, Polycoma, Rhizodiscina, Saccharata, Aaosphaeria, Amniculicola, Byssothecium, Aspergillus, Meira, Trypanosporium, Lizonia, Aureobasidium, Morchella, Sodiomyces, Spermosporium, Jaminaea, Ceraceosorus, Testicularia, Tilletiopsis, Violaceomyces, Rhizopus, Alternaria, Hesseltinella, Neurospora, Cylindrosporium, and Rhizosporium.

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