In vitro bioproduction of polyalkanoates from polypropylene and polyethylene

Abstract

An enzymatic method and system for producing a bioplastic polymer from a used product containing a thermoplastic polymer is disclosed. The thermoplastic polymer-containing product after use can be pyrolyzed to obtain a combined liquor of depolymerized alkanes. The combined liquid of alkanes may be contacted in vitro with an enzyme or a mixture of enzymes to produce a bioplastic polymer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 411,657, filed on October 15, 2019, which is expressly incorporated herein by reference in its entirety. Background Art

[0003] It is estimated that the output of petroleum-based polymers exceeds 300 million tons per year, and global output continues to increase. Most of the 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. With the worsening of the spread of disposable plastics around the world, it is crucial to identify fully renewable plastics and develop methods and materials for industrial processing of renewable plastics.

[0004] Biodegradable polymers (also called "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). Much work has been done on the PHA family, most notably polyhydroxybutyrate (PHB) polymers, including poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO), and their copolymers. Of particular advantage, PHAs exhibit very similar thermoplastic properties to some petroleum-based polymers and therefore represent a viable alternative to petroleum-based polymers such as polypropylene and polyethylene. However, currently, PHA synthesis, whether natural or otherwise, is limited to C1-C8 PHAs, which has hampered research on PHAs (i.e., 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. 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 catalytic formation of β-ketoacyl-CoA from acetyl-CoA by PhaA (β-ketothiolase). In turn, it is converted to R-3-hydroxyacyl-CoA by the PhaB enzyme (β-ketoacyl-CoA reductase) in a NADP-dependent reaction. The final step is the polymerization of R-3-hydroxyacyl-CoA into PHB catalyzed 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. Biosynthesized polyhydroxyalkanoates accumulate in bacterial cells as large molecular weight particles that can account for about 60% to about 90% of the cell dry matter. Therefore, it would be advantageous if petroleum-based precursors could be used for microbially mediated PHA synthesis.

[0006] However, while PHA products biodegrade significantly faster than petroleum-based polymers, it has proven very difficult to utilize petroleum-based precursors to form bioplastics. That is, while microorganisms should theoretically be able to degrade petroleum-based products into usable precursors, the high molecular weight of petroleum-based products such as polyethylene (PE) and polypropylene (PP) inhibits or even prevents microbial mediation.

[0007] Therefore, there is a need for systems and methods that can completely convert biopolymers from petroleum-based precursors in vitro. Bioplastics can enzymatically break down petroleum-based polymers into monomer units, and then use the monomers to produce new polymers. The true recycling of such bioplastics will bring significant progress to waste treatment processes. If the recovered and reformed biopolymers are suitable for consumer products and industrial processes, it will be another beneficial effect. Additionally or alternatively, it is economically and environmentally advantageous to enzymatically obtain bioplastic polymers using alkanes obtained from post-consumer products containing petroleum-based thermoplastics. Providing an in vitro enzymatic process for forming bioplastic polymers from post-consumer products containing thermoplastics will be an additional benefit. Summary of the invention

[0008] Generally speaking, the present disclosure relates to methods and systems for producing bioplastic polymers from post-consumer products containing petroleum-based thermoplastic polymers.

[0009] Post-consumer products containing petroleum-based thermoplastic polymers may include components of 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 containing petroleum-based thermoplastic polymers may also include components of packaging, post-industrial use, and / or other polymer wastes. The present disclosure relates to a method that can be used for the combination of single-system biodegradation and the formation of new biopolymers in small or large environments.

[0010] In one aspect, the present disclosure generally relates to an enzymatic method for producing a bioplastic polymer from a post-consumer product containing a petroleum-based thermoplastic polymer, which enzymatic method can include pyrolyzing the post-consumer product containing a petroleum-based thermoplastic polymer to obtain a combined liquid of depolymerized alkanes. The combined liquid of alkanes can be contacted in vitro with an enzyme or enzyme mixture to produce a bioplastic polymer.

[0011] In one aspect, the method can include a post-use product containing a petroleum-based thermoplastic polymer, and the post-use product containing a petroleum-based thermoplastic polymer includes polypropylene and / or polyethylene. In another aspect, the method can have a combined liquid of an alkane of 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. In an exemplary aspect, one or more steps of the method are carried out together in one container or more than one container. In an exemplary aspect, the method can be carried out in a temperature range of about 40°C to about 80°C.

[0012] In another aspect, the method may include one or more additional steps: for each of the one or more additional steps, the amalgam of alkanes is contacted with an enzyme or enzyme mixture in vitro by repeating the enzymatic step. Such one or more additional steps may be repeated at least three times. For example, the method may include contacting the amalgam of alkanes with a first enzyme in vitro, then contacting one or more subsequent enzymes in a stepwise manner, or contacting the amalgam of alkanes with two or more enzymes in vitro simultaneously. The method may include at least one additional enzyme different from the first enzyme.

[0013] In one aspect, the method can produce a polyalkanoate bioplastic polymer. The polyalkanoate produced can be a polyhydroxyalkanoate and / or a polyhydroxybutyrate. In one aspect, the polyhydroxyalkanoate produced can be characterized as reacting chemically in a manner comparable to that of polypropylene or polyethylene. In another aspect, the polyhydroxyalkanoate 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. In one aspect, the bioplastic polymer produced can have a linear carbon chain, can be at least about 80% homogeneous, can have a mass yield proportional to the optical density measurement obtained, and / or can contain substantially minimal amounts of any microplastics and / or nanoplastics. For example, containing substantially minimal amounts of any microplastics and / or nanoplastics may include microplastics and / or nanoplastics in an amount of about 0.01% to about 10% of the total mass yield.

[0014] In another aspect, the method can include an enzyme or enzyme mixture purified from an extremophile 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, Met hyloligella, Micromonospora, Natronococcus, Nocardiopsis, Paracoccus, Roseivax, Saccharomonospora, Shewanella, Alicyclobacillus, Natranaerobius, Halobacteriaceae, Hyphomonas, Amycolatopsis, Georgenia, Acidothermus, Thermobifida, or a combination thereof. The microorganism can be an engineered microorganism that has been genetically modified to secrete a specific enzyme for use in the method. The microorganism can be at least one type of naturally occurring microorganism that naturally encodes a specific enzyme for use in the method. In one aspect, the enzyme or enzyme mixture can be a thermophilic enzyme. Thermophilic enzymes can tolerate temperatures from about 40°C to about 120°C.

[0015] In one aspect, the method may include at least one enzyme purified from Lihuaxuella thermophila. In another aspect, the method may include enzymes that effectively synergize in the same environment, the same environment being characterized by identical or similar pH and temperature.

[0016] In another aspect, the present disclosure also relates generally to a method for producing bioplastic polymers from alkanes without organisms. For example, the method may include contacting one or more alkanes 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 a linear bioplastic polymer. The linear bioplastic polymer may include polyhydroxyalkanoates having a carbon chain length greater than C8.

[0017] In yet another aspect, the present disclosure also generally relates to uncharacterized polyhydroxyalkanoates. For example, the uncharacterized polyhydroxyalkanoates can have a carbon chain length greater than C8 and / or can be a linear polymer substantially devoid of any side chain pendant polymers.

[0018] In another aspect, the present disclosure also generally relates to a system configured for simultaneous biodegradation of post-consumer products and production of polyhydroxyalkanoates. For example, the system may include one or more containers configured to keep a combined solution of alkanes obtained from pyrolysis of post-consumer products in contact with a purified enzyme or purified enzyme mixture.

[0019] In yet another aspect, the present disclosure is also generally directed to a method for producing polyalkanoates in a multi-step enzymatic reaction, which can include contacting a combined liquid of alkanes with an alkane monooxygenase in vitro to obtain an alcohol. For example, the method can include contacting an alcohol with an alcohol dehydrogenase to obtain an aldehyde, contacting an aldehyde with an aldehyde dehydrogenase 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 a polyhydroxyalkanoate, 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 synthase to obtain polyhydroxybutyrate. For example, long-chain fatty acid CoA ligase / synthetase can be purified from Thermobifida halotolerans. The alcohol dehydrogenase can be a fungal long-chain alcohol dehydrogenase purified from the group of fungal genera including: Pullulan, Venturia, Lophium, Tothia, Trichodelitschia, Westerdykella, Sphaerotheca, Viridothelium, Cryptosporidium, Zopfia, Polycoma, Rhizodiscina, Saccharata, Aaosphaeria, Amniculicola, Byssothecium, Aspergillus, Meira, Triatomine, Lizonia, Pullulan, Morchella, Sodiomyces, Spermobacterium, Jaminaea, Ceraceosorus, Testicularia, Tilletiopsis, Violaceomyces, Rhizopus, Alternaria, Hesseltinella, Neurospora, Cylindrosporium and Rhynchosporium.

[0020] Other features and aspects of the disclosure are discussed in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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:

[0022] Figure 1 is a graphic representation of the cleavage of carbon-carbon bonds based on alkane reactions, which can be metabolized through a series of consecutive hydroxylation reactions of PP or PE, which ultimately produces acetate that enters the TCA cycle and can produce microplastics and nanoplastics;

[0023] Figure 2 is a schematic representation of the biochemical basis for the conversion of PP / PE into new biomaterials while avoiding the formation of microplastics and other direct polymer decomposition products. The dashed arrows indicate the various steps;

[0024] Figure 3 is a diagram of the conversion of long-chain fatty acid acyl-CoA to PHA and acetyl-CoA;

[0025] Figure 4 yes Figure 3 Schematic representation of the family of conversion of the produced acetyl-CoA to PHB and the use of two molecules of long-chain acyl-CoA to produce PHA.

[0026] FIG. 5A(i) is Figure 2 Enzymes used in reaction 1: sequences of alkane monooxygenase EC 1.14.15.3;

[0027] Figure 5A(ii) is Figure 2 Enzyme used in reaction 2: sequence of alcohol dehydrogenase EC1.1.1.2;

[0028] Figure 5A(iii) is Figure 2 Enzymes used in reaction 3: Sequence of aldehyde dehydrogenase EC1.2.5.2;

[0029] Figure 5A(iv) is Figure 2 Enzymes used in reaction 4: sequences of long-chain fatty acid CoA ligase / synthetase EC6.2.1.3;

[0030] FIG. 5B(i) is Figure 3 Enzymes used in reaction 1: Sequence of long-chain acyl-CoA dehydrogenase EC 1.3.8.8;

[0031] Figure 5B(ii) is Figure 3 Enzymes used in reaction 2: Sequence of long-chain enoyl-CoA hydratase EC4.2.1.17;

[0032] Figure 5B(iii) is Figure 3 Enzymes used in reaction 3: sequences of EC 1.1.1.211 / 1.1.1.35;

[0033] Figure 5B(iv) is Figure 3 Enzymes used in reaction 4: sequence of hydroxyacyl-CoA dehydrogenase EC 1.1.1.36;

[0034] Figure 5B(v) is Figure 3 Enzymes used in reaction 5: sequences of poly(R)-hydroxyalkanoate synthase EC 2.3.1.304;

[0035] FIG. 5B (vi) is Figure 3Enzymes used in reaction 6: Sequence of acetyl-CoA C-acyltransferase EC 2.3.1.16;

[0036] Figure 5C(i) is Figure 4 Enzymes used in reaction 1: Sequence of acetoacetyl-CoA synthase EC 2.3.1.9;

[0037] Figure 5C (ii) is the sequence of PHB depolymerase EC3.1.1.75;

[0038] Figure 5C (iii) is the sequence of a fungal long-chain fatty alcohol dehydrogenase;

[0039] Figure 5C(iv) is a diagram that can be Figure 3 The sequence of the trifunctional enzyme that replaces reaction 2 and reaction 3 in ;

[0040] Figure 6 is a graphical representation of the reaction catalyzed by long-chain fatty acid CoA synthetase / ligase used to monitor the overall progress of the reaction;

[0041] Figure 7 is through Figure 2 Graphical representation of the formation of long chain fatty acid CoA measured by pyrophosphate formation in the terminal reaction;

[0042] Fig. 8A is a graphical representation of the production of long-chain fatty acid CoA as a function of the amount of crude bacterial extract added to the reaction;

[0043] Figure 8B is a graphical representation of the production of long chain fatty acid CoA as a function of the amount of alkane slurry added to the reaction;

[0044] Fig. 9 It is a graphical representation of the formation of PHA over time;

[0045] Fig.10 is a graphical representation of the depolymerization of PHA over time by purified X. thermophila PHA depolymerase.

[0046] 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

[0047] definition

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Detailed Description

[0054] 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.

[0055] In general, the present disclosure relates to an enzymatic method for producing bioplastic polymers from post-consumer products containing petroleum polymers. Unexpectedly, the enzymatic method of the present disclosure can produce PHB and PHA bioplastic polymers by using olefins produced from the pyrolysis of polypropylene and polyethylene. For example, in an exemplary aspect, the method of the present disclosure depolymerizes petroleum-based polymers to provide a combined liquid of alkanes produced by, for example, pyrolysis, which can be used in an in vitro enzymatic method to provide various polyalkanoates (including PHB and PHA). That is, the present disclosure has unexpectedly found that by carefully selecting a combination of enzymes and process conditions, novel PHA polymers of different chain lengths can be produced without the need for multi-step or multi-container methods, and even in vivo methods that limit the production of biopolymers and the production rate of biopolymers can be avoided.

[0056] For example, in one aspect, an enzymatic method for producing a bioplastic polymer from a post-consumer product containing a petroleum-based thermoplastic polymer may include a) pyrolyzing the post-consumer product containing a petroleum-based thermoplastic polymer, b) obtaining a combined liquid of depolymerized alkanes from the pyrolyzed post-consumer product, c) contacting the combined liquid of alkanes with an enzyme or enzyme mixture in vitro, and d) producing a bioplastic polymer. For example, the post-consumer product containing a petroleum-based thermoplastic polymer may include components of 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). The post-consumer product containing a petroleum-based thermoplastic polymer may also include components of industrial post-consumer and / or other polymer wastes.

[0057] In yet another 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, and these pollutants include but are not limited to mesophilic pathogens, such as but not limited to viruses, bacteria, fungi and protozoa, and these pollutants can eliminate pathogenicity by the disclosed method. As used herein, the term "mesophilic" ("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 / 1000 to about 18 / 1000 (also referred to as medium salt), about an atmospheric pressure (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.

[0058] However, no matter what kind of decontamination is needed, the disclosure as a whole is all related to enzyme or enzyme mixture (especially enzyme or enzyme mixture selected for carrying out one or more reactions with alkane monomers, as discussed in more detail below) and use rear product to be combined.For example, in an example, enzyme can be combined with use rear product, and this use rear product contains the incontinence product discarded or other polymer-based consumer goods made of petroleum-based thermoplastic polymer, such as food container, beverage container, packaging etc.Incontinence product comprises for example diaper, training pants, swimming trunks, adult incontinence product, feminine hygiene product etc.These products generally comprise permeable liner, outer cover and absorbent structure positioned between liquid permeable liner and outer cover.Incontinence product can contain the amount greater than about 5 % by weight, such as the amount greater than about 10 % by weight, such as the amount greater than about 20 % by weight, such as the amount greater than about 30 % by weight, such as the amount greater than about 40 % by weight, such as the amount greater than about 50 % by weight, such as the amount greater than about 60 % by weight, such as the petroleum-based thermoplastic polymer greater than about 70 % by weight.

[0059] As described above, in one aspect, the enzymatic methods outlined herein can recycle post-consumer products containing polypropylene and polyethylene to produce bioplastic polymers. This allows post-consumer products to be recycled because such products made from petroleum-based polymers can be broken down to enzymatically produce new (fully recycled) bioplastic polymers. That is, the present disclosure has discovered that by utilizing pyrolysis to release alkanes from petroleum-based polymers, a unique combination of enzymes can be selected to produce post-consumer products that can then be converted into bioplastic polymers.

[0060] For example, a post-consumer product containing a petroleum-based thermoplastic polymer may contain polypropylene and / or polyethylene. Without wishing to be bound by theory, it is believed that the cleavage of carbon-carbon bonds based on alkane reactions can be metabolized through a series of sequential hydroxylation reactions of petroleum-based polymers (such as PP or PE), which ultimately produce acetate to enter the TCA cycle (e.g. Figure 1 In addition, it is believed that this mechanism can avoid the production of microplastics and nanoplastics, which is an additional benefit compared to previous chemical degradation methods of petroleum-based polymers, as nanoplastics and microplastics are of increasing concern because they may be more toxic than intact petroleum-based polymers.

[0061] For example, disclosed herein is an overall metabolic pathway that begins with the pyrolysis of a pool of alkanes from one or more petroleum-based polymers (such as, in one example, PP and / or PE). Pyrolysis of one or more petroleum-based polymers (such as, in one example, PP and / or PE) can produce a distribution of alkanes (e.g., C6-C12 (PP 15 and PE 33, by way of example only), C13-C16 (PP 33 and PE 31, by way of example only), C17-C20 (PP 13 and PE 14, by way of example only), and C20-C30 (PP 25 and PE 12, by way of example only)). For example, many of these long chain carbons can be used in the methods of the present disclosure to produce PHAs of known and / or new chain lengths. In addition, the entire pyrolysis pool can be used as Figure 2 Inputs to the method shown. In addition, as described above, it should be understood that addition petroleum-based polymers can be utilized, as well as petroleum-based polymers of varying lengths.

[0062] Subsequently, the combined stream of alkanes from the pyrolysis of PP and / or PE can be converted to long-chain primary alcohols, long-chain aldehydes, long-chain fatty acids (LCFA), and ultimately to long-chain fatty acid coenzyme A (LCFA CoA) molecular populations (see Figure 2 ). LCFA CoA can serve as the main metabolic entry point for the production of PHA.

[0063] Once a pool of LCFA-CoA molecules is produced, the LCFA-CoA molecules need to be converted to their cognate PHAs. This can be done according to the present disclosure. Figure 3 In one aspect, Figure 2 and Figure 3 The reactions can be run together or separately depending on the design of the overall process or by careful selection of enzymes. For example, the LCFA CoA is converted to long chain acetoacetyl-CoA in three steps. Depending on the choice of enzymes added to the reaction, this molecule can have two different metabolic pathways. For example, if a hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36) can be used to form a pool of hydroxyacyl-CoA, the hydroxyacyl-CoA can polymerize into a family of PHAs and release CoA, which can then be Figure 2 , and then reused in the last reaction in order to reform the LCFA CoA molecule. Alternatively, a combined solution of acetyl-CoA and long-chain acyl-CoA molecules can be produced using, for example, acetyl-CoA C-acyltransferase (EC 2.3.1.16). Figure 4 , these two molecules can be used to synthesize the PHB and / or PHA family.

[0064] In one aspect of the methods disclosed herein, the combined liquid of alkanes can include any one or more alkanes 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.

[0065] In another aspect, steps c) and d) 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, steps c) and d) of the present disclosure can be performed together in one container or in more than one container.

[0066] 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, selecting 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 an alkane pool. 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 2 Any source of enzyme in the reaction.

[0067] Thus, in one aspect, the method may include a step of contacting the combined solution of alkanes 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 do not utilize Figures 2 to 4 The enzyme of any intermediate produced during the reaction, the method starting from the amalgam of alkane discussed herein can be carried out as a "one-pot method" reaction, allowing further improvement in efficiency, speed and floor space. However, in one aspect, the method can include one or more additional steps: for each in one or more additional steps, by repeating step c), the amalgam of alkane is contacted with enzyme or enzyme mixture in vitro. For example, one or more additional 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 additional 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.

[0068] In addition, in another aspect, the method can include contacting the combined solution of alkanes with a first enzyme in vitro, and then contacting one or more subsequent enzymes in a stepwise manner. For example, the combined solution of alkanes can be contacted with a first enzyme in vitro, and then contacted with a second enzyme, and then contacted with one or more optional subsequent enzymes. In yet another aspect, the method can include contacting the combined solution of alkanes with two or more enzymes simultaneously in vitro.

[0069] 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 include some enzymes that are different from each other, and some enzymes can be identical or similar. For example, one or more subsequent enzymes can include at least one additional enzyme that is different from the first enzyme.

[0070] 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 inventors have 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 bioplastic polymers. For example, engineered enzyme variants can also be adapted to specific reactions or overall reaction processes.

[0071] In one aspect, the enzyme or enzyme mixture in step c) can be purified from an extremophile 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, Met hyloligella, Micromonospora, Natronococcus, Nocardiopsis, Paracoccus, Roseivax, Saccharomonospora, Shewanella, Alicyclobacillus, Natranaerobius, Halobacteriaceae, Hyphomonas, Amycolatopsis, Georgenia, Acidothermus, Thermobifida, or a combination thereof.

[0072] In one aspect, the enzyme or enzyme mixture in step c) can 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 can 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), Minwuiathermotolerans(WP-206420073), Rhodopseudomonas thermotolerans(WP-114356866), Rhodopseudomonas pentothenatexigens(WP-114356866), Streptomyces thermotolerans(WP-114356866), 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), Thermoflavimicrobium dichotomicum)(WP-093229000), Thermogemmatispora carboxidivorans(WP-081839208), Thermogemmatispora aurantia(WP-151728970), Thermogemmatispora tikiterensis(WP-11243376), Thermogemmatispora onikobensis(WP-084659191), Thermoleophilaceae bacteria(MBA2429278), Thermomonosporaechinospora(WP-160147065), Thermomonosporacellulosilytica(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 bacteria. .

[0073] 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 unusual isoelectric points, not requiring enhanced biosafety measures, not containing excess cysteine ​​residues, being generally non-in vivo, being commercially available, or a combination thereof, which will be discussed in more detail below.

[0074] However, although enzymes present in solution have been discussed so far, it should be understood 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 the specific enzyme used in step c).

[0075] In addition to the microorganisms that naturally express a certain enzyme gene, one or more genetically modified bacteria can also be selected, and the bacteria express an exogenous enzyme that can carry out the specific reaction of the present invention. In addition, the microorganism can be an engineered microorganism that has been genetically modified to secrete the specific enzyme used in step c).

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.).

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] The following genera may also be selected to express the enzymes of the present invention according to the present disclosure:

[0092] Firmicutes: Bacillus, Xulihua, and Clostridium;

[0093] Proteobacteria: Bradyrhizobium, Sphingomonas, Azotobacter, Azospirillum, Nitrobacter, Lysobacter, Stenotrophomonas, Rhizobium, Acinetobacter, Thiobacillus, Schlegelella, Janthinobacterium, Sinorhizobium, Pseudomonas, Agrobacterium, and Escherichia (e.g., Escherichia coli);

[0094] Actinobacteria: Rhodococcus, Arthobacter, Streptomyces, Conexibacter, Rhodococcus, Solirubrobacter, Micrococcus, Rubrobacter, and Actinomyces;

[0095] Bacteroidetes: Flavobacterium and Pedobacter;

[0096] Deinococcus-thermus: Deinococcus and Thermus;

[0097] Gemmatimonads: Gemmatimonas and Gemmatironas;

[0098] Spirochaetes: Tumeriella and Leptospira;

[0099] Verrucomicrobia: Pedosphaera, Chthoniobacter and Verrucomicrobia;

[0100] Chloroflexi: Thermogemmatispora and Dictyobacter; and

[0101] Armatimonadetes: Fimbriimonas

[0102] 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.

[0103] 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, Eutropha 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, Xanthomonas vesicatoria 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.

[0104] In another aspect, step c) of the method can include enzymes that effectively act synergistically in the same environment, 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.

[0105] In some aspects, fungal enzymes can be used for the methods of the present disclosure. For example, fungal long-chain fatty 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 is lowered. For example, Figure 5C (iii) provides examples of fungal enzymes that can be utilized.

[0106] 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.

[0107] In other aspects, the bioplastic polymer produced in step d) of the method can be 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 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.

[0108] In one aspect, the bioplastic polymer produced in step d) can have a linear carbon chain. In another aspect, the bioplastic polymer produced in step d) 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 bioplastic polymer produced is directly related to the chain length of the alkanes contained in the combined liquid of alkanes.

[0109] In another aspect, the mass yield of the bioplastic polymer produced in step d) can be proportional to the optical density measurement obtained. For example, the higher the optical density reading, the higher the mass yield of the bioplastic polymer produced. In yet another aspect, the bioplastic polymer produced in step d) can contain substantially the lowest amount of any microplastics and / or nanoplastics. For example, any microplastics and / or nanoplastics containing substantially the lowest amount 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. 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 bioplastic polymer produced in an undetectable amount.

[0110] In certain aspects, the bioplastic polymer produced 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)).

[0111] 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.

[0112] In other aspects, the present disclosure relates to a biomass-free method for producing bioplastic polymers from alkanes. For example, a combined solution of depolymerized alkanes can be contacted with a purified enzyme or a purified enzyme mixture in vitro to produce a bioplastic polymer. For example, the enzymatic reaction of the biomass-free method can be carried out in a single container or in more than one container. In addition, the biomass-free method can be carried out at a temperature range of about 40°C to about 80°C, or any variant thereof as described above.

[0113] In one aspect, the abiotic method can include one or more additional steps: for each of the one or more additional steps, the amalgam of alkanes is contacted with an enzyme or an enzyme mixture 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 the amalgam of alkanes with a first enzyme in vitro, then contacting with one or more subsequent enzymes in a stepwise manner, or the abiotic method can include contacting the amalgam of alkanes 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 amalgam of alkanes and all necessary enzymes contacted 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.

[0114] In another aspect, the bioplastic polymer produced in the biomass-free method can include polyalkanoates. For example, the polyalkanoate can be polyhydroxyalkanoate and / or polyhydroxybutyrate. The polyhydroxyalkanoate can be characterized as chemically reacting in a manner comparable to that of polypropylene or polyethylene. The polyhydroxyalkanoate 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.

[0115] In one aspect, the bioplastic polymer produced in the biofree process can include linear carbon chains. In one aspect, the bioplastic polymer produced in the biofree process can be at least 80% homogeneous, or any variation thereof as discussed above. In another aspect, the mass yield of the bioplastic polymer produced in the biofree process can be proportional to the optical density measurement obtained. In another aspect, the bioplastic polymer produced in the biofree process can contain substantially minimal amounts of any microplastics and / or nanoplastics.

[0116] 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.

[0117] In another aspect, the organism-free method may include contacting one or more alkanes 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 a linear bioplastic polymer. For example, the linear bioplastic polymer may include polyhydroxyalkanoates having a carbon chain length greater than C8 and / or a carbon chain length less than C30.

[0118] 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.

[0119] In one aspect, the present disclosure generally relates to a system configured for simultaneous biodegradation of post-consumer products and production of polyhydroxyalkanoates. For example, the system may include one or more containers configured to keep a combined solution of alkanes obtained from pyrolysis of post-consumer products in contact with a purified enzyme or purified enzyme mixture.

[0120] In another aspect, the present disclosure generally relates to a method for producing polyalkanoates in a multi-step enzymatic reaction. For example, the method can include contacting a combined solution of alkanes in vitro with an alkane monooxygenase to obtain an alcohol, contacting the alcohol with an alcohol dehydrogenase to obtain an aldehyde, contacting the aldehyde with an aldehyde dehydrogenase to obtain a long-chain fatty acid, contacting the 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 contacting with a long-chain enoyl-CoA hydratase, then contacting with a hydroxyacyl-CoA dehydrogenase to obtain a long-chain acetoacetyl-CoA, and contacting the long-chain acetoacetyl-CoA with a hydroxyacyl-CoA dehydrogenase to obtain a hydroxyacyl-CoA for polymerization into a polyhydroxyalkanoate, or

[0121] Long chain acetoacetyl-CoA is contacted with an acetyl-CoA C-acyltransferase to obtain acetyl-CoA, and acetyl-CoA is contacted with an acetoacetyl-CoA synthase to obtain a polyhydroxyalkanoate (such as PHB in this example). Of course, as described above, in one aspect, each of the enzymes mentioned above can be added simultaneously. That is, as discussed, by carefully selecting compatible enzymes, such as those discussed above, a one-pot reaction is possible, allowing all enzymes to be added simultaneously, so that the reaction proceeds naturally from the combined solution of alkanes to produce PHA 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 with reference to the following examples.

[0126] In a specific embodiment, a crude extract of the bacterium Thermospora fusca (ATCC-27730) was prepared by sonication on ice. The released cytoplasm contained all the required enzymes and cofactors at initial concentrations. In addition, an expressed form of long-chain fatty acid CoA synthetase / ligase was added to the crude extract of Thermospora fusca to help drive the reaction forward and provide an easily monitored reaction to evaluate the reaction.

[0127] A typical reaction contained (in 1.0 mL final volume): varying amounts of crude bacterial extract, 20 mM ATP, 5 mM MgCl2, 5 mM CaCl2, 5 mM KCl, 20 mM CoA-sodium salt, varying amounts of long-chain fatty acid CoA synthetase / ligase, and varying amounts of alkane pool from polyethylene pyrolysis. The reactions were incubated at 50°C. The long-chain fatty acid CoA ( Figure 2 The final reaction product in the pathway and Figure 3 pathway) to determine Figure 2 Whether the method steps are completed. In other words, Figure 6 Specific reactions catalyzed by long-chain fatty acid CoA synthetase / ligase are provided for monitoring the overall progress of the reaction.

[0128] The generation of pyrophosphate (PPi) was monitored by spectroscopy. The formation of PPi was determined using a MAK168 fluorescence-based kit from Millipore-Sigma Chemical Co. At time points, aliquots were taken from the reaction and mixed with a fluorescence assay reagent. Samples were excited at 316 nm and the fluorescence emission intensity was measured at 456 nm in a Molecular Devices SpectraMax M5.

[0129] from Figure 7 As can be seen in the figure, under complete reaction conditions, after a lag period, the fluorescence emission intensity increases over time (solid circles). The reaction shows some effect of intrinsic PPi-degrading enzyme activity, as the curve begins to decrease after 150 minutes. The plateau reached by the reaction likely represents Figure 2 Depletion of a key cofactor somewhere in the reaction pathway. If exogenous long-chain fatty acid CoA synthetase / ligase is not added in the reaction, the overall rate of LCFA CoA is linear, but still measurable, and the amount of the final product is significantly reduced (open circles). If exogenous CoA, long-chain fatty acid CoA synthetase / ligase and ATP are not added in the reaction, PPi (solid squares) exceeding the basal level will not be formed. Therefore, all reactants and enzymes are added to the crude extract, and a reaction force that drives the total reaction toward the direction of forming long-chain fatty acid CoA can be formed. Finally, the amount of long-chain fatty acid CoA that can be produced in the standard reaction depends on the amount of the added alkane combined liquid starting material, as shown in Figure 8.

[0130] After the 300 minute incubation period, the expanded reaction was centrifuged at 10,000 x g for 20 minutes to precipitate any debris. The aqueous phase was decanted into a clean tube. Fresh crude cell extracts of Geobacillus thermoleovorans (ATCC BAA898) bacteria were added to the reaction containing additional 10 mM MgCl2, 10 mM CaCl2, 10 mM KCl, varying amounts of long-chain enoyl-CoA hydratase (EC 4.2.1.17) and varying amounts of PHA polymerase (EC 2.3.1.34). The reaction was incubated at 45°C and the progress of the reaction was monitored over time by measuring the increase in optical density at 650 nm.

[0131] As PHA is formed in the reaction, it precipitates as a light gray material that increases light scattering. Again, all cofactors and enzymes required for the reaction are contained in the crude bacterial extract, and the two purified enzymes help drive the entire reaction in the forward direction. After a short hysteresis period (approximately 40 minutes), material begins to precipitate from the reaction solution, and material continues to form during the reaction. A plateau begins to appear at approximately 140 minutes (solid circles). Using only the crude extract of Bacillus thermophilus bacteria for the reaction resulted in a moderate increase in optical density (open circles). In the absence of bacterial extract, no change in optical density was observed (solid squares).

[0132] After the enzymatic reaction was complete, the material was mixed with an equal volume of 70°C chloroform for one hour with gentle stirring. The chloroform layer was separated from the aqueous phase and poured into a glass petri dish to a depth of approximately 2 mm and the solvent was allowed to evaporate at 25°C. The samples were aged for five days (1.0 atm, 25°C) and then vacuum dried for 3 hours to remove any residual chloroform. The dried material was scraped into a cuvette and mixed with a solution of 10 mM MgCl2, 10 mM CaCl2, 10 mM KCl, 5 mM CHES (pH 9.0) and 1 mg / mL Thermophilic Xulihua PHB depolymerase. The reaction was incubated at 70°C with stirring while measuring the optical density of the solution at 650 nm. The time course of the reaction was 240 °C. Fig.10 As shown in . PHA material solubilizes with enzymatic hydrolysis. However, about 25% of the material is still insoluble. Therefore, PHA is formed by the above method as shown by the degradation of PHB depolymerase.

[0133] The sequences of the various enzymes used in the examples are Figures 5A(i) to 5C(iv) Available in.

[0134] This is embodied in a multi-step enzymatic process that produces PHB and a variety of PHA bioplastic polymers from alkanes obtained from the pyrolysis of PP and / or PE. Thus, this example demonstrates the steps of pyrolyzing a post-consumer product containing a petroleum-based thermoplastic polymer to obtain a pool of alkanes, and then enzymatically converting the pool of alkanes into PHA monomers suitable for the production of bioplastic polymers.

[0135] 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 interchanged 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 process for producing a bioplastic polymer from a post-consumer product containing a petroleum-based thermoplastic polymer, the enzymatic process comprising: a) pyrolyzing the post-consumer product containing a petroleum-based thermoplastic polymer; b) obtaining a combined liquid of depolymerized alkanes from the pyrolyzed spent products; c) contacting the combined solution of alkanes with an enzyme or enzyme mixture in vitro, and d) Producing bioplastic polymers.

2. The method of any one of the preceding claims, wherein the after-use product containing a petroleum-based thermoplastic polymer comprises polypropylene and / or polyethylene.

3. A method as described in any of the preceding claims, wherein the combined liquid of alkanes comprises any one or more alkanes 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.

4. The method according to any one of the preceding claims, wherein step c) and step d) are performed together in one container.

5. A method as claimed in any one of the preceding claims, wherein steps c) and d) are carried out in more than one vessel.

6. The method according to any one of the preceding claims, wherein the method comprises one or more further steps: for each of the one or more further steps, the combined solution of alkanes is contacted in vitro with the enzyme or the enzyme mixture by repeating step c).

7. The method of claim 6, wherein the one or more additional steps are repeated at least three times.

8. A method as claimed in any one of the preceding claims, wherein the method comprises contacting the pool of alkanes in vitro with a first enzyme and then with one or more subsequent enzymes in a stepwise manner.

9. The method of claim 8, wherein the one or more subsequent enzymes include at least one additional enzyme different from the first enzyme.

10. A method as claimed in any one of the preceding claims, wherein the method comprises contacting the pool of alkanes simultaneously with two or more enzymes in vitro.

11. A method as claimed in any one of the preceding claims, wherein the bioplastic polymer produced in step d) is a polyalkanoate.

12. The method of claim 11, wherein the polyalkanoate is a polyhydroxyalkanoate.

13. The method of claim 12, wherein the polyhydroxyalkanoate is characterized as reacting chemically in a manner comparable to that of polypropylene or polyethylene.

14. The method of claim 12, wherein the polyhydroxyalkanoate has 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.

15. The method of claim 11, wherein the polyalkanoate is polyhydroxybutyrate.

16. A method as claimed in any one of the preceding claims, wherein the bioplastic polymer produced in step d) has a linear carbon chain.

17. A method as described in any of the preceding claims, wherein the bioplastic polymer produced in step d) is at least about 80% homogeneous.

18. A method as claimed in any one of the preceding claims, wherein the mass yield of the bioplastic polymer produced in step d) is proportional to the optical density measurement obtained.

19. A method as claimed in any one of the preceding claims, wherein the bioplastic polymer produced in step d) contains substantially minimal amounts of any microplastics and / or nanoplastics.

20. The method of any one of the preceding claims, wherein the enzyme or the enzyme mixture in step c) is purified from an extremophile microorganism.

21. The method of claim 20, wherein the microorganism is a bacterium of the genus Halomonas, Xulihuatella, Lysobacter, Alteromonas, Arthrobacter, Azospirillum, Steadybacter, Desulfovibrio, Halobacillus, Halobacterium, Halobacterium, Halomasinicola, Halorhodomyces, Halorhodomyces, Halorhodomyces, Halorhodomyces, Halorhodomyces, Halobacterium, Halobacter, Termitomyces, Marinobacter, Methyloligella, Micromonospora, Haloalkalicoccus, Nocardia, Paracoccus, Roseochromia, Saccharomonas, Shewanella, Acidobacillus, Alkalianaerobe, Halobacteraceae, Mycena, Amycolatopsis, Georgesella, Thermoacidobacter, Thermosporium, or a combination thereof.

22. The method of claim 20, wherein the microorganism is an engineered microorganism that has been genetically modified to secrete a specific enzyme for use in step c).

23. The method of claim 20, wherein the microorganism is at least one type of naturally occurring microorganism that naturally encodes a specific enzyme used in step c).

24. The method of any one of the preceding claims, wherein the enzyme or the enzyme mixture in step c) is a thermophilic enzyme.

25. The method of claim 24, wherein the thermophilic enzyme is resistant to temperatures of about 40°C to about 120°C.

26. The method of any one of the preceding claims, wherein step c) and step d) are performed at a temperature in the range of about 40°C to about 80°C.

27. A method as claimed in any one of the preceding claims, wherein at least one enzyme in step c) is purified from X. thermophila.

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

29. An abiotic process for producing a bioplastic polymer from an alkane, the abiotic process comprising: contacting one or more alkanes with the purified enzyme or purified enzyme mixture in an environment substantially absent of any bacteria secreting the same enzyme or the same enzyme mixture; and Produces linear bioplastic polymers.

30. A method as claimed in claim 29, wherein the linear bioplastic polymer comprises a polyhydroxyalkanoate having a carbon chain length greater than C8.

31. An uncharacterized polyhydroxyalkanoate, the uncharacterized polyhydroxyalkanoate having: A carbon chain length greater than C8, wherein the polyhydroxyalkanoate is a linear polymer substantially devoid of any side chain pendant polymer groups.

32. A system constructed for simultaneous biodegradation of post-consumer products and production of polyhydroxyalkanoates, the system comprising one or more containers configured to maintain a combined liquid of alkanes obtained from pyrolysis of post-consumer products in contact with a purified enzyme or a purified enzyme mixture.

33. A method for producing polyalkanoate by a multi-step enzymatic reaction, the method comprising: contacting the pool of alkanes with an alkane monooxygenase in vitro to obtain an alcohol; contacting the alcohol with an alcohol dehydrogenase to obtain an aldehyde; contacting the aldehyde with an aldehyde dehydrogenase to obtain a long-chain fatty acid; contacting the 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 to obtain a long-chain acetoacetyl-CoA; and contacting a long chain acetoacetyl-CoA with a hydroxyacyl-CoA dehydrogenase to obtain hydroxyacyl-CoA for polymerization into polyhydroxyalkanoate; or Long-chain acetoacetyl-CoA is contacted with an acetyl-CoA C-acyltransferase to obtain acetyl-CoA, and the acetyl-CoA is contacted with an acetoacetyl-CoA synthase to obtain polyhydroxybutyrate.

34. The method of claim 33, wherein the long-chain fatty acid CoA ligase / synthetase is purified from Thermospora halophila.

35. The method of claim 33, wherein the alcohol dehydrogenase is a fungal long-chain alcohol dehydrogenase purified from the group of fungal genera comprising Aureobasidium, Venturia, Lophium, Tothia, Trichodelitschia, Westerdykella, Sphaerotheca, Viridothelium, Cryptosphaeria, Zopfia, Polycoma, Rhizodiscina, Saccharata, Aaosphaeria, Amniculicola, Byssothecium, Aspergillus, Meira, Triatomyces, Lizonia, Aureobasidium, Morchella, Sodiomyces, Spermosporium, Jaminaea, Ceraceosorus, Testicularia, Tilletiopsis, Violaceomyces, Rhizopus, Alternaria, Hesseltinella, Neurospora, Cylindrosporium, and Rhizosporium.

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