Patent Application
20240150803
The XML file USC0336PUSP Sequences.xml of size 72,592 created Oct. 15, 2023, filed herewith, is hereby incorporated by reference.
In at least one aspect, the present invention is related to the conversion of waste plastics to high-value metabolites.
Plastic production is currently accelerating at a rate faster than any other material on the planet [1, 2], and is estimated to reach a global production volume of 1.1 billion tons annually by 2040. Only 9% of plastics were recycled as of 2015 (3). Millions of tons of plastics, in the form of trillions of plastic particles [4] leak from waste management systems into the environment, posing increasing threats to the food supply and ecosystems [5]. Polyesters are frequently recycled (ca. 30% of polyethylene terephthalate (PET)), unlike polyolefins (ca. 6% of low-density polyethylene (LDPE)) [2, 3]. Due to their robust microstructures and excellent physicochemical properties, polyethylenes have been utilized to deliver countless improvements to quality of life and health. Polyethylenes will remain ubiquitous and thus it is desirable master reclamation of the value embedded in these materials at the end of their lives.
The same physicochemical properties that make plastics useful also prevent their degradation and recycling. Further exacerbating this problem are the additives that necessarily accompany any post-consumer waste stream, e.g. colorings and plasticizers. Unlike polyesters and nylons, the chemical methods known to recycle or remanufacture polyethylenes are usually harsh [6, 7, 8]. Separately, oxidant-free, catalytic approaches are emerging for polyethylene upcycling [9, 10]. While these have modest yields and require energy-intensive conditions, they avoid the potential uncontrolled reactions that can result from heating organics with O2. Still, oxidative conditions have an important advantage of tolerance to impurities associated with post-consumer polymer waste. These concerns, particularly salts, are exacerbated in samples recovered from the oceans or recycling centers.
Chemical approaches to polyethylene degradation generate a diverse distribution of products, because there are no functional handles in their pure hydrocarbon structures to direct a from cleavage either limits the value of these products or creates a challenge of separating them. Thus, there is growing interest in employing biological systems to break down plastics. Enzymatic degradation [11, 12] and microbial cell factories [13] have both shown moderate success in the degradation of certain plastics, but these efforts focus on degradation rather than value reclamation. Routes to upcycle plastics to secondary metabolites are not known, despite the enormous value creation that such a path would enable. Robust synthetic biology approaches have not yet been effectively developed to overcome existing limitations in plastic upcycling.
Accordingly, there is a need for improved methods for converting plastics to high-value metabolites.
In at least one aspect, a non-human organism for upgrading intermediate oxidation products formed by catalytic degradation of polyethylenes, polypropylenes, or polystyrenes is provided. The non-human organism is genetically modified to the intermediate oxidation products to secondary metabolites, and in particular to include a positive feedback loop construction in the promotor system.
In another aspect, a method for upgrading intermediate oxidation products formed by catalytic degradation of alkanes or polystyrenes is provided. The method includes steps of catalytically degrading linear and/or branched alkanes or polypropylene in an oxidizing environment to form intermediate oxidation products with a catalyst system that includes one or more catalysts and contacting the intermediate oxidation products with a non-human organism. Advantageously, the non-human organism is genetically modified to convert the intermediate oxidation products to secondary metabolites.
In another aspect, a method for making a biocontrol agent is provided. The method includes steps of culturing a strain of Aspergillus flavus with benzoic acid in a culture medium and collecting spores from the strain of Aspergillus flavus therefrom.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein:
Reference will now be made in detail to presently preferred compositions, embodiments and methods of the present invention, which constitute the best modes of practicing the invention presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the invention and/or as a representative basis for teaching one skilled in the art to variously employ the present invention.
Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word “about” in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary: percent, “parts of,” and ratio values are by weight; the term “polymer” includes “oligomer,” “copolymer,” “terpolymer,” and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.
It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and/or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.
It must also be noted that, as used in the specification and the appended claims, the singular form “a,” “an,” and “the” comprise plural referents unless the context clearly indicates otherwise. For example, reference to a component in the singular is intended to comprise a plurality of components.
The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.
The phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
The phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4 . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits. In the specific examples set forth herein, concentrations, temperature, and reaction conditions (e.g. pressure, pH, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to three significant figures. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to three significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pH, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to three significant figures of the value provided in the examples.
In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.
The term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. A “tissue specific” promoter is only active in specific types of tissues or cells.
To determine the “percent identity” (i.e., percent sequence identity) of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a refinement, the sequences are aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. In a refinement, the length of a first sequence aligned for comparison purposes is at least 80% of the length of a second sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. In this regard, the following oligonucleotide alignment algorithms may be used: BLAST (GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: BLASTN; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http://www.embl-heidelberg.de/Services/index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters. When sequences differ in conservative substitutions, the percent identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
“AUG” means area under the curve.
“BGC” means biosynthetic gene clusters.
“DMAPP” means dimethylallyl pyrophosphate.
“FPP” means farnesyl pyrophosphate.
“FRP” means fiber-reinforced polymer.
“GMM” means glucose minimal media.
“GPP” means geranyl pyrophosphate.
“HDPE” means low-density polyethylene.
“IPP” means isopentenyl pyrophosphate.
“LDPE” means low-density polyethylene.
“LEL” means lower explosivity limit.
“NHPI” means N-hydroxyphthalimide.
“NO” means nitric oxide.
“PHA” means polyhydroxyalkanoate.
“PMM” means polyethylene minimal media.
“PPMM” means polypropylene minimal medium.
“PP” means polypropylene.
“PS” means polystyrene.
“PSMM” means PS-derived BA.
“SM” means secondary metabolite.
“TPP” means tetraphenylporphyrin.
“UEL” means upper explosivity limit.
The following sequences are provided with respect to the modified fungi set forth below: SEQ ID NO: 1 (afoA (AN1029) (Aspergillus nidulans)); SEQ ID NO: 2 (afoD (AN1033) (Aspergillus nidulans)); SEQ ID NO: 3 (alcR (AN8978) (Aspergillus nidulans); SEQ ID NO: 4 (ctvA (Aspergillus terreus)); SEQ ID NO: 5 (ctvB (Aspergillus terreus)); SEQ ID NO: 6 (ctvC (Aspergillus terreus); SEQ ID NO: 7 (ctvD (Aspergillus terreus)); SEQ ID NO: 8 (pl-ggs (Clitopilus passeckerianus)); SEQ ID NO: 9 (pl-cyc (Clitopilus passeckerianus)); SEQ ID NO: 10 (pl-p450-1 (Clitopilus passeckerianus)); SEQ ID NO: 11 (pl-p450-2 (Clitopilus passeckerianus)); SEQ ID NO: 12 (pl-sdr (Clitopilus passeckerianus)); SEQ ID NO: 13 (Pl-p450-3 (Clitopilus passeckerianus)); SEQ ID NO: 14 (Pl-atf (Clitopilus passeckerianus)); SEQ ID NO: 15 (AN7620 (egt1) (Aspergillus nidulans)); SEQ ID NO: 16 (AN6227 (egt2) (Aspergillus nidulans)); SEQ ID NO: 17 (Afu2g15650 (Aspergillus fumigatus)); SEQ ID NO: 18 (Afu2g13295 (Aspergillus fumigatus)); SEQ ID NO: 19 (NCU04343 (Neurospora crassa)); SEQ ID NO: 20 (NCU11365 (Neurospora crassa)) and SEQ ID NO: 21 (AN3307 (agsB) (Aspergillus nidulans)). In some refinements, nucleotide sequences having at least (in increasing order of preference) 70, 80, 90, 95, 97 or 99 percent identity to SEQ ID NOS 1-10 can be used.
In an embodiment, a non-human organism for upgrading intermediate oxidation products formed by catalytic degradation of alkanes or polystyrenes is provided. Characteristically, the non-human organism is genetically modified to convert alkanes and/or polystyrene oxidation products to secondary metabolites.
In another aspect, the alkanes are linear and/or branched alkanes. In a refinement, the alkanes are C8-50 linear and/or branched alkanes. In a refinement, linear and/or branched alkanes, are polyethylenes or polypropylenes, or mixtures thereof. In another refinement, the linear and/or branched alkanes are provided as new or used motor oil-based materials.
In another aspect, the alkanes are polyethylene. In a refinement, the alkane is a low-density polyethylene having a weight average molecular weight from about 20,000 to 100,000 g/mol. In another refinement, the alkane is a high-density polyethylene having a weight average molecular weight from about 100,000 to 1,000,000 g/mol. In another refinement, the alkane is a linear low-density polyethylene having a weight average molecular weight from about 30,000 to 500,000 g/mol.
In another aspect, the alkanes are polypropylene. In a refinement, the alkane is a low-density polypropylene having a weight average molecular weight from about 50,000 to 500,000 g/mol.
In another aspect, the polystyrene has a weight average molecular weight from about 100,000 to 300,000 g/mol.
In a variation, the intermediate oxidation products include C10-25 aldehydes, C10-25 esters, C10-25 compounds having ring (i.e., fused or unfused) systems, C8-50 linear and/or branched alkanes, C4-20 Carboxylic acids or dicarboxylic acids, or combinations thereof In a refinement, the secondary metabolites include ergothionine. In a further refinement, the secondary metabolites include asperbenzaldehyde. In another refinement, the secondary metabolites include citreoviridin and mutilin.
In a variation, the non-human organism is a fungus. In a refinement, the non-human organism is Aspergillus nidulans or Aspergillus flavus.
In a variation, the non-human organism is genetically modified by removing the afoD gene. Details of nucleotide sequences for modified (i.e., knockout) Aspergillus nidulans are found in U.S. Pat. No. 10,118,945; the entire disclosure of which is hereby incorporated by reference.
In another variation, the non-human organism is genetically modified by replacing the promoter of the afoA promoter with the alcA promoter (alcA(p)) in the nuclear genome.
In another variation, the non-human organism is genetically modified by replacing the promoter of the alcR gene with the constitutive gpdA promoter in the nuclear genome.
In another variation, the non-human organism is genetically modified by removing the afoD gene, replacing the promoter of the afoA gene with the gpdA promoter and inserting an additional copy of the afoA gene under control of the afoE promoter in the nuclear genome. This creates a positive feedback loop that generates high levels of both AfoA (protein) and asperbenzaldehyde.
In another variation, the non-human organism is genetically modified by deleting the entire sterigmatocystin biosynthetic gene cluster (genes stcA-stcW) and the emericellamide biosynthetic gene cluster (genes easA-easD) in the nuclear genome. (see, SEQ ID NO: 1 from U.S. Pat. No. 10,118,945; the entire disclosure of which is hereby incorporated by reference).
In another variation, each heterologously expressed gene in the non-human organism is placed under control of alcA(p) in the nuclear genome.
In another variation, the non-human organism is genetically modified by amplifying genes AN7620 and AN6227 using A. nidulans genomic DNA and inserting AN7620 and AN6227 replacing the coding regions of afoG and afoF, respectively and replacing the native promoter of afoA with alcA(p) to create strain YM267 and wherein maintenance of the native promoters of each of these genes allows for the protein AfoA to bind to native promoters and drive expression of the genes AN7620 and AN6227. In a refinement, the coding regions of afoE and afoD are replaced with the A. fumigatus egt1 (Afu2g15650) and egt2 (Afu2g13295) homologs, respectively, to yield strain YM812. In a further refinement, a third pair of ergothioneine biosynthetic genes are inserted into the regulon by replacing the coding regions of afoC and afoB with the N. crassa egt1 (NCU04343) and egt2 (NCU11365) genes to yield strain YM4820. In a further refinement, deleting the agsB gene encoding an α-1,3-glucan synthase is deleted to create strain YM4847.
In another embodiment, a method for upgrading intermediate oxidation products formed by catalytic degradation of degrading linear and/or branched alkanes or polystyrenes is provided. The method includes steps of catalytically degrading linear and/or branched alkanes or polypropylene in an oxidizing environment to form intermediate products with a catalyst system that includes one or more catalysts, and contacting the intermediate products with a non-human organism. Advantageously, the non-human organism is genetically modified to convert the intermediate oxidation products to secondary metabolites.
In a variation, the one or more catalysts include a transition metal-containing catalyst. The one or more catalysts may include MeReO3 and oxides and halides of Co, Mn, Cu, and Re (e.g., Co(II), Mn(II), Cu(I), Re(VII), Re(V), and Re(III)). In a refinement, the one or more catalysts include a hydroxylated amine (e.g. N-hydroxyphthalimide). In another refinement, the one or more catalysts include NO. In another refinement, the one or more catalysts include Fe(acac)2 or Fe(acac)3. It should be appreciated that any combination of these catalysts can be used.
In another aspect of the method, the catalyst system includes a cocatalyst. In a refinement, the cocatalyst includes hydroxylated amines. For example, the cocatalyst can include hydroxysuccinamide (NHS) or hydroxylamine. The co-catalyst can also include N-hydroxyphthalimide (NHPI). In another refinement, the co-catalyst includes include NO. In a refinement, the linear and/or branched alkanes, are polyethylenes or polypropylenes, or mixtures thereof. In a particularly useful example, branched alkanes are provided as motor oil.
In another aspect of the method, the intermediate oxidation products include C10-25 aldehydes, C10-25 esters, C10-25 compounds having imbedded ring systems, benzoic acid, C4-20 carboxylic acids or dicarboxylic acids, thereof.
In another aspect of the method as set forth above, the secondary metabolites include ergothionine. In a refinement, the secondary metabolites include asperbenzaldehyde. In a refinement, the secondary metabolites include citreoviridin and mutilin.
In another aspect of the method as set forth above, the non-human organism a fungus, and in particular, a genetically modified fungus. Details of the genetically modified fungus are set forth above and in the experimental section below.
In another aspect, a method for making a biocontrol agent is provided. The method includes steps of culturing a strain of Aspergillus flavus with benzoic acid in a culture medium; and collecting spores of the strain of Aspergillus flavus therefrom. In a refinement, the strain of Aspergillus flavus is A. flavus Af36.
In another aspect, the benzoic acid is formed by catalytically degrading a polystyrene in an oxidizing environment to form intermediate products with a catalyst system that includes one or more catalysts. Details of the catalyst system including the one or more catalysts and the co-catalysts are set forth above.
In another aspect, a method for making a biocontrol agent is provided. The method includes steps of culturing B. bassiana GHA in a culture medium that includes polypropylene digestion products and collecting spores from the B. bassiana GHA therefrom. The polypropylene digestion products can be formed by catalytically degrading a polypropylene in an oxidizing environment to form intermediate products with a catalyst system that includes one or more catalysts. Details of the catalyst system including the one or more catalysts and the co-catalysts are set forth above. In a refinement, the culture medium that includes polypropylene digestion products can be formed by adding these digestion products to a MM. In a refinement, GMM, and MM can be used as controls.
Additional details of the embodiments set forth herein are found in C. Rabot et al. Conversion of Polyethylenes into Fungal Secondary Metabolites (First published: 3 Nov. 2022), Volume 62, Issue 4, Jan. 23, 2023 https://doi.org/10.1002/anie.202214609; Am. Chem. Soc. 2023, 145, 9, 5222-5230, Feb. 13, 2023, https://doi.org/10.1021/jacs.2c12285 and supplemental materials; C. Rabot et al., Polystyrene Upcycling into Fungal Natural Products and a Biocontrol Agent, J. Am. Chem. Soc. 2023, 145, 9, 5222-5230, Publication Date: Feb. 13, 2023 https://doi.org/10.1021/jacs.2c12285 and supplemental materials; and in U.S. Pat. No. 10,118,945; the entire disclosures of which are hereby incorporated by reference in their entireties.
The following examples illustrate the various embodiments of the present invention. Those skilled in the art will recognize many variations that are within the spirit of the present invention and scope of the claims.
In this section, we aimed to exploit fungi, which produce products worth billions of dollars each year[14] to biologically upcycle polyethylenes. Their biosynthetic products include medically valuable secondary metabolites (SMs) including antibiotics, the cholesterol-lowering statins, immunosuppressants, and antifungals. [15] Because they have been reported to use diacids as carbon sources,[16,17] we sought to generate structurally diverse and pharmacologically active SMs directly from polyethylene-derived substrates.
It is shown here that post-consumer polyethylenes can be rapidly degraded to generate substrates that are suitable for upgrading by fungal metabolism. As a proof of principle, we demonstrate that these plastic-derived substrates can be used to produce the diverse SMs asperbenzaldehyde, citreoviridin, and mutilin in useful yields (Scheme 1). We also demonstrate robust genetic engineering strategies that permit the expression of biosynthetic gene clusters (BGCs) from many different organisms. Thus, in principle, this method expands the catalog of products to which polyethylenes can be upcycled to thousands of SMs.
By adapting conditions for the conversion of cyclohexane to adipic acid, [18] we were able to optimize an initial system for polymer cleavage. Using O2 consumption and 1H NMR integration for indicative signals as our characterization handles (by 1H NMR), we eventually found conditions based on cobalt and manganese salts and a phthalamide-based NO source[8] that give useful oxidative cleavage results (Table 1.1). The distribution of α,ω-diacid products that are produced by the oxidative chemistry was further quantified by GCMS.
We observed that re-charging our reactor with additional O2 did not restart the polymer cleavage reaction and hypothesized that N-hydroxyphthalimide (NHPI) serves as a source of NO, which is vented from the reactor headspace upon O2 recharge. We see rapid hydrolysis of NHPI to phthalic acid upon reaction initiation. We further observed that our metallic catalysts lost reactivity in the recharge process (Table 1.3, entries 4-6), and that a better result was obtained when metal salts were added portion-wise along with O2 and NHPI at recharge (compare entries 6-7). Under conditions optimized for full polymer conversion to relatively small diacid products (
Its highly tolerant O2-based conditions give this method the critical and distinguishing feature of tolerance of post-consumer wastes. We demonstrate that feature here with four examples (
Fungi represent attractive candidates for diacid upgrading due to their robust growth capabilities, inexpensive cultivation requirements, engineerable metabolic pathways, and potential to synthesize metabolites with potent and diverse bioactivities. Short chain diacids, however, have been reported to inhibit fungal growth. [19] We confirmed that C4-C8 (photographs not shown) were toxic to the model filamentous fungus A. nidulans (strain FGSC A4) even when glucose was present as a carbon source. We found, however, that A. nidulans utilizes C10 and C12 diacids as sole carbon sources (photographs not shown) without signs of toxicity. We thus devised a system to separate polyethylene digestion products of ≥10 carbons from those <10 carbons. A series of pH-controlled liquid-liquid extractions permitted the rapid separation of C10+ diacids from light diacids and metal salts (
In a representative example (vide supra), 27 wt % of ocean-sourced polyethylenes were converted to diacids that were discretely identifiable using authentic standards. It should be noted that light diacids are not waste products. They may be used in large-market applications such as in the synthesis of PBCx, a biodegradable plastic emerging in agricultural applications. [20] Our data also suggest that these light diacids possess antifungal properties (photographs not shown) that may be exploited.
For attempts to produce SMs from polyethylene-derived diacids, the heavy diacid extract was added to liquid minimal media at a concentration of 10 g L−1. Liquid cultures were inoculated with fungal strains and incubated for several days (see ESI for a full extraction protocol, culture conditions, and media recipes). SMs were analyzed and quantified from culture extracts via HPLC-DAD and HPLC-DAD-MS.
Initial attempts to elicit SM production from various wild-type fungal strains resulted in only small amounts of SMs as detected via HPLC-DAD-MS. We consequently genetically engineered A. nidulans to overexpress SM biosynthetic genes or biosynthetic gene clusters (BGCs) and this proved effective, allowing robust and efficient SM production.
In order to determine the versatility of this system, we attempted to engineer fungal strains to produce various SMs using several BGC activation/expression approaches (Table 1.5). The SM used as a readout for the first of these systems was asperbenzaldehyde, a major polyketide intermediate in asperfuranone biosynthesis. [21] Asperbenzaldehyde and its derivatives disassemble tau filaments, inhibit lipoxygenases, and inhibit the interactions of the oncogenic RNA-binding proteins HuR and Musashi-1 with their target mRNAs. [22-24] We chose to target a biosynthetic intermediate because it can serve as a discovery platform that can easily be synthetically modified.
passeckerianus origin)
We developed three strains with different systems for driving asperbenzaldehyde production: LO2955, LO8355, and LO10050. All molecular genetic modifications were executed using previously described fusion PCR-based construct generation and transformation protocols. [25] In strain LO2955, the afoD gene was deleted, blocking asperfuranone biosynthesis such that asperbenzaldehyde, its biosynthetic precursor, accumulates. Further, the promoter of the afoA gene that encodes the transcription factor (AfoA) that drives expression of the asperfuranone BGC was replaced with the alcA promoter (alcA(p)), which is highly inducible with a variety of alcohols and ketones, including methyl ethyl ketone. [26] To increase expression of AfoA, we next replaced the promoter of the alcR gene with the strong, constitutive gpdA promoter[27] in LO2955, creating strain LO8355. The alcR sequence encodes a transcription factor that drives expression of alcA. [28]
In addition, we developed a new, strong constitutive promoter system that employs a positive feedback loop (
Yields of each strain grown in liquid lactose minimal media (LMM) were quantified via HPLC-DAD (
To determine the general utility of the system, we also attempted to express the diterpene antibiotic platform mutilin from the basidiomycete Clitopilus passeckerianus and the F1-ATPase β-subunit inhibitor citreoviridin from A. terreus var. aureus. [29] Mutilin is an intermediate in the biosynthetic pathway for pleuromutilin, which binds to the peptidyl transferase center of the bacterial ribosome, thus halting protein synthesis. [30] Mutilin is therefore an attractive platform for medicinal discovery efforts toward overcoming bacterial antibiotic resistance. Further, basidiomycetes are phylogenetically distant from ascomycetes such as A. nidulans and the ability to produce mutilin would indicate that this system works for BGCs from very diverse fungi. Citreoviridin is a potent mycotoxin that uncompetitively and noncompetitively inhibits ATP hydrolysis and ATP synthesis, respectively, by binding to the β-subunit of F1-ATPase. [31] Compounds in this class of mycotoxins have been investigated for the treatment of cancer. [32] In total, four genes from A. terreus var. aureus and five genes from C. passeckerianus were transferred into an A. nidulans recipient strain and placed under control of alcA(p) to generate robust producers of citreoviridin and mutilin, respectively.
Engineered fungal strains were incubated in liquid minimal media supplemented with 10 g L−1 polyethylene digest extracts (MINI, polyethylene minimal media). Culture media and/or mycelia were extracted with appropriate organic solvents, which were then analyzed via HPLC-DAD or HPLC-DAD-MS (
Our results indicate that engineered fungal strains can efficiently produce useful quantities of each target SM in under one week. Interestingly, microscopic examination of LO10050 when cultured in liquid PMM revealed initial stunted growth relative to GMM controls (by Phase contrast micrographs of asperbenzaldehyde-producing strain LO10050 in GMM and PMM, not shown). However, we observed ample hyphal growth after 48 hours and abundant asperbenzaldehyde crystals after 72 hours of incubation in PMM, which is consistent with our findings regarding asperbenzaldehyde titers.
These yields are in contrast to other metabolic engineering efforts; while high-yielding strains have been reported following extensive engineering, [33] ample SM production typically requires much larger quantities of carbon source(s) to achieve comparable yields. [34-36] It is also noteworthy that our yields were obtained from shake flasks with minimal optimization. Alteration of other culture parameters known to influence fermentation titers (e.g. culture length, media components, etc.) should permit significantly higher yields. Use of the strong constitutive promoter system may increase the production of citreoviridin and mutilin and codon optimization may further increase mutilin production.
We further note that it was not necessary to employ metabolic engineering strategies to confer the ability to metabolize polymer-derived diacids to the fungi; rather, simple extraction protocols selectively isolated diacids suitable for fungal metabolism. Finally, it is quite likely that polyethylene degradation products can be used as a carbon source in the production of other SMs. The BGCs that we have expressed are from diverse fungi and the approaches we have developed should permit the expression of BGCs from many sources. The combination of the catalytic degradation of polyethylenes with the genetic engineering of filamentous fungi represents a promising strategy for plastic upcycling.
We present a method to rapidly upcycle post-consumer polyethylenes into structurally diverse and medically useful SMs. We degrade these polyethylenes using oxidative catalysis to generate a distribution of diacids. These diacids are rapidly isolated and upgraded by engineered strains of A. nidulans to synthesize bioactive SMs. Taken together, this two-step process dramatically expands the catalog of products to which polyethylenes can be upcycled to thousands of SMs.
We initiated our investigation of conditions for PS degradation using Styrofoam insulated boxes (Table 2.1) that we shredded by hand. We monitored BA generation via 1H NMR integration as a readout for the optimization of our catalytic conditions. Low conversion efficiencies were observed with manganese(II) acetoacetonate, while the corresponding nitrate salt afforded a more reactive cleavage system (entries 1-3). Interestingly, we found that the introduction of cobalt together with manganese synergistically promoted BA generation. For example, 5 wt % each of two nitrate salts afforded a 26% yield of BA after four hours, but 10 wt % of either catalyst alone could not permit a comparable conversion (entries 3-5).
Based on these results, we hypothesize that manganese is likely to act as a Lewis acid in catalyzing electron-transfer oxidation from tertiary carbons in the polymer backbone, and cobalt tends to catalyze the β-scission for C—C bond cleavage[37]. Furthermore, under optimal conditions with portion-wise recharging of O2, we observed the complete degradation of PS with up to 71% of starting polymer recovered as BA and 84% total mass recovery (entries 5-7). The added balance comprises incompletely digested oligomers. We also note that a reduction in reaction time did not afford increased product yield, even under optimized conditions (entries 8-10).
We evaluated our conditions on four additional post-consumer PS sources: a Styrofoam plate, waste collected from Santa Catalina Island, CA, a disposable coffee cup lid, and a red drink cup (
aStyrofoam cold box;
bStyrofoam plate;
cCatalina Island waste;
dCoffee lid;
eRed drink cup.
Following catalysis optimization, we developed a simple procedure to isolate polymer digest for downstream fungal upgrading. A series of liquid-liquid extractions followed by recrystallization afforded BA in high purity as indicated by 1H NMR. This isolated BA was then used for downstream metabolomics experiments. For a detailed extraction protocol, see Materials & Methods.
We first confirmed that fungi can utilize BA as a sole carbon source using the model filamentous fungus A. nidulans FGSC A4 (photographs, not shown). We observed a slight discoloration in the presence of BA relative to glucose minimal media (GMM) positive controls, indicating some degree of BA-induced toxicity. We separately determined that BA was not significantly toxic to the fungus. We repeated the above experiments with phthalic acid, the hydrolysis product of the NHPI catalyst, to confirm that it is both unable to be metabolized and is nontoxic to the fungus.
For initial metabolomics experiments, we utilized the strain A. nidulans LO10050 to determine if SMs can be generated from a BA standard as a carbon source. As reported previously, [38] LO10050 has been engineered to express certain genes from the asperfuranone biosynthetic gene cluster (BGC). The incorporation of a positive feedback promoter system into this strain drives the production of the biosynthetic intermediate asperbenzaldehyde to very high levels. The high production yield of asperbenzaldehyde allowed us to easily compare initial culture conditions permissive of SM production from BA.
We took advantage of this robust asperbenzaldehyde production system to determine preliminary culture conditions for the fungal metabolism of PS-derived BA. We noticed that incubation of fungal strains in the presence of a BA standard affected the morphology of the strain; spherical mycelia characteristic of filamentous fungi cultured in shake flasks were not observed in these culture conditions. Nevertheless, a microscopic examination of the culture medium revealed the presence of extensive hyphae, revealing that growth attributable to BA was still occurring. We therefore chose to use asperbenzaldehyde production as a surrogate endpoint for fungal growth during these initial experiments.
Due to its reported toxicity to fungi [39], we next determined the concentration of a BA standard that permitted the highest yield of asperbenzaldehyde in liquid cultures. We found that LO10050 could dose-dependently utilize BA as a sole carbon source to generate asperbenzaldehyde (
To promote the efficient bioconversion of waste-derived BA in high yield, several genetic engineering strategies were employed to generate three medically—and industrially relevant SMs: ergothioneine, pleuromutilin, and mutilin. The first SM that we aimed to generate from BA was ergothioneine, an unusual thio-histidine betaine amino acid. [40] Ergothioneine is a natural antioxidant that can be microbially synthesized by certain species of fungi and actinobacteria. It has been reported to exhibit anti-inflammatory and cytoprotective properties, leading to its growing application in the pharmaceutical and cosmetic industries. [41]
Although discovered more than a century ago, there has recently been exponential growth in publications related to ergothioneine. [42] The genetic basis of its biosynthesis has been elucidated both in fungi and actinobacteria. In the latter, five genes (egtABCDE) direct its biosynthesis from histidine and cysteine. [43] However, fungi can synthesize ergothioneine using only two genes: egt1 and egt2.48,49 To engineer a strain to produce ergothioneine in useful quantities, we took advantage of the robust A. nidulans afo regulon[44] to express ergothioneine biosynthetic genes. This regulon natively governs the production of the polyketide SM asperfuranone; overexpression of afoA, encoding a cluster-specific transcription factor, has been shown strongly to activate all genes within the BGC, leading to the production of very high levels of products of the cluster. To exploit the robust expression profile of this regulon, we replaced the coding regions of genes within the BGC with genes involved in ergothioneine biosynthesis.
First, BLASTp was used to identify putative A. nidulans homologs using the Neurospora crassa egt1 and egt2 as queries. This recovered two sequences bearing moderate homology to egt1 and egt2: AN7620 (63% similarity/50% identity) and AN6227 (58% similarity/43% identity). AN7620 and AN6227 were then amplified using A. nidulans genomic DNA and inserted into the coding regions of afoG and afoF, respectively. Maintenance of the native promoters of each of these genes allows for afoA to bind to them and drive their expression. We additionally replaced the native promoter of afoA with alcA(p) to create strain YM267.
To further increase yields, we replaced the coding regions of afoE and afoD with the A. fumigatus egt1 (Afu2g15650) and egt2 (Afu2g13295) homologs, respectively, to yield strain YM812. We inserted a third pair of ergothioneine biosynthetic genes into the regulon by replacing the coding regions of afoC and afoB with the N. crassa egt1 (NCU04343) and egt2 (NCU11365) genes to yield strain YM820. Finally, we deleted the agsB gene encoding an α-1,3-glucan synthase[45] to create strain YM847. Deletion of genes encoding α-1,3-glucan synthases has been shown to improve fermentation titers by reducing hyphal clumping when grown in liquid cultures. [46] Collectively, these genetic engineering strategies enabled the generation of a strain expressing an inducible system controlling three pairs of ergothioneine biosynthetic genes (
We next utilized strains of A. nidulans engineered to synthesize the SMs pleuromutilin and mutilin from BA. Pleuromutilin, a diterpene natural product produced by the basidiomycete Clitopilus passeckerianus, was first discovered in 1950[47]. It and its derivatives function by selectively inhibiting bacterial translation by binding to the peptidyl transferase center of the bacterial ribosome. [48, 49] Recently, the semisynthetic pleuromutilin derivative lefamulin was approved by the FDA for the treatment of community-acquired bacterial pneumonia. [50] Its biosynthetic pathway, involving seven genes in total, was elucidated in 2017. [51] We utilized strain YM343, reported recently by our group, [52] that was engineered to reconstitute the entire pleuromutilin biosynthetic pathway to produce pleuromutilin from PS-derived BA. Each gene of interest was placed under control of the afo regulon to drive expression to very high levels. We also utilized strain YM283, which expresses only five of the seven genes within the pleuromutilin BGC, to produce its precursor, mutilin. Production of a biosynthetic precursor of the final product in the pathway should enable late-stage synthetic derivatization. Details regarding the biosynthetic pathway of mutilin and pleuromutilin are shown in
We next determined if engineered fungal strains can biosynthesize SMs from PS-derived BA. Metabolic profiling of strains YM847, YM283, and YM343 revealed that all three metabolites can be produced in useful quantities from engineered strains of A. nidulans when grown in minimal media with PS-derived BA (PSMM) as a carbon source (
Finally, we sought to determine if spores of an agriculturally-relevant biocontrol agent can be generated from PS-derived BA. This is of particular importance to the field of plastics upcycling, because the biocatalytic products produced to date, while more valuable than the parent polymer, are not frequently used on scales that approach the quantity of plastic that will need to be reclaimed. Thus, it is valuable to add widely-used agrichemicals to our product portfolio. To determine feasibility of this application, we cultured A. flavus Af36 on solid GMM and PSMM agar plates and quantified spore generation after a seven-day incubation period. Our results indicate that spores of A. flavus Af36 can readily be generated using PS-derived BA (
Building out a robust and diversified portfolio of methods to reclaim value embedded in waste plastics, including PS, remains an evergreen challenge. Here, we couple catalytic oxidation with genetic engineering to develop a route to convert PS to fungal SMs within one week. In principle, any SM for which the biosynthetic pathway has been elucidated should be able to be generated from these PS-derived substrates, provided a robust expression system is established. We also demonstrate that we can generate spores of the agriculturally relevant biocontrol agent A. flavus Af36 from this PS-derived substrate. This adds important depth to the biocatalytic plastics upcycling portfolio because it shows a route that would consume upcycled plastics on an agrichemical scale.
Further, the enzymatic nature of biosynthesis coupled with high SM yields described here and previously[53] also implies high protein levels. Thus, this approach should also enable the production of proteins themselves from these plastic-derived substrates. Lastly, the production of other fungal fermentation products, such as organic acids, dyes, biofuels, and biopolymers should be available by these same methods. Thus, this platform effectively expands the catalog of products derived from PS from a few, structurally simple compounds to, in principle, thousands of natural products. It opens the path to apply material upcycled from ocean-sourced waste to the promotion of human health and protection of the global food supply.
The following experiments show that (1) used motor oil can be valorized through a route analogous to the route introduced for LDPE, and that (2) ligated complexes of cobalt, manganese, iron, or copper, derived from the salts that were used on LDPE, will affect the analogous cleavage of hydrocarbon waste materials with some selectivity for larger molecular weight cleavage fragments than observed for the parent salts. This is possible because the bulk of a supporting ligand will direct oxidation by the metal to peripheral regions of a folded hydrocarbon molecule, enabling valorization of lower molecular weight waste hydrocarbons like used motor oil.
It is demonstrated that the methods described above can process motor oil into substrates that can be recycled using fungal metabolism, establishing a baseline efficiency for our method.
The polymer cleavage chemistry set forth above can be adapted to smaller molecular weight hydrocarbon feedstocks, particularly used motor oil, which requires the development of a more end-chain-selective oxidation catalyst. To explain the problem, this section will provide background on what motor oil is and what our new chemistry must accomplish.
Engine oils and light lubricants primarily comprise C18-050 alkanes of irregular branching. [54] Some oils (American Petroleum Institute (API) Groups I-III) are natural distillates used directly. Some oils and additives comprise upgraded (usually hydrotreated) natural oils (Group IV) and synthetic poly(alpha-olefins) (Group V). Commercial engine oils also contain pH buffers, polyols, detergents, and even sulfur (1%), zinc (700 ppm), and phosphorus compounds. At the point of disposal, used engine oil contains all of these materials, plus metal particles, soot, PCBs (1 ppm), lead (16 ppm), and thermally- and oxidatively-degraded fragments from the oil itself. [55] Thus, our chemical system must tolerate all of these materials and separate or destroy any that are toxic to fungi.
When the polyethylene methodology was designed, it was envisioned that a linear polyethylene would be stochastically cleaved into linear diacids, and these would be recognized by fungi as if they were fatty acids. It was observed that some of our polymer digest materials are inert (will not support or inhibit growth, like unfunctionalized alkanes) or toxic (prevent growth in glucose media) to the primary host organism, Aspergillus nidulans. It was confirmed that C4-C8 (studied individually at 1/5/10 g/L)2 are toxic to A. nidulans strain FGSC A4 with glucose present as a carbon source. However, A. nidulans utilizes C10 and C12 diacids as sole carbon sources without signs of toxicity.
The synthetic blend (Groups I-IV) new motor oil of
Fungi grow on motor oil digest. When our most prolific asperbenzaldehyde producer strain of A. nidulans (LO10050, vide infra) is grown on the motor oil digest of
The degree of branching in motor oil digest could be problematic for fungal growth, it is anticipated that this will be tolerated, as in LLDPE. The conversion of postconsumer LL/LDPE samples into fungal metabolites have been shown without observable difference in the efficiency of the fungal upgrading step compared to HDPE. We record higher yields for chemical cleavage of HDPE than LDPEs, which we attribute to branches in the polymer, not because they are not tolerated, but because we do not have analytical standards to quantify them: we pass these fragments on to fungal upgrading without quantification. Together, these observations teach us that branched fragments in LDPE are tolerated in our oxidative cleavage chemistry and that their oxidized products are substrates for fungal metabolism.
Higher branching density challenges the organism's tolerance. When PP is treated with our oxidative conditions (Table 3.1), we see hydroxylation of the polymer's tertiary centers: 1H NMR shows the absence of carbinol protons, but alcohol carbons are present in 13C NMR and HMBC. We suspect that tertiary alcohols are disadvantageous for fungal metabolism. HMBC further shows a preponderance of carbonyl signals appropriate for carboxylates and none for ketones, so we are confident that we are making diacids. LC/MS and 1H DOSY data for PP digest show fragment sizes similar to PEs. We have not yet realized high yields of medicinal products from PP digest, but we have produced triacetic acid lactone, a bulk chemical, and industrially relevant biocontrol agents AF36.2 A high degree of branching reduces the effectiveness of these materials as fungus food, but they are metabolized. The level of branching in motor oil digest should be between those in LDPE and PP.
It has been shown that the addition of a bidentate nitrogen ligand (bipy, phenanthroline, TMEDA, or the Tilset MesN═C(Me)—C(Me)═NMes diamine) will halt polyethylene cleavage under our optimized conditions: bidentate N—N ligands kill the catalyst(s). Addition of tetraphenylporphyrin (TPP) does not retard reactivity in this way: in a pair of experiments, we digested 1 g motor oil using Mn(NO3)2+Co(NO3)2 (2.5 wt % each) with NHPI (10 wt %) under O2 (6 atm, HOAc, 150° C.) and the presence and absence of TPP (10 wt %). The reactions proceed at similar conversion (per near-identical O2 consumption), indicating that the porphyrin does not significantly stop or accelerate reactivity, possibly because it does not ligate the metal(s). Our proposal requires that the ligand should modify oxidation selectivity, which is unlikely if it does not bind a metal. We probed this through 1H NMR integration (
To generate spores of B. bassiana GHA biocontrol agents, six-well cell culture plates (VWR; r=1.75 cm) containing 10 mL solid GMM, PPMNI, or MM were prepared. 1.0×106 spores of B. bassiana GHA were inoculated into each well, spread using a plastic spreader, and incubated for seven days at 30° C. Following incubation, 3 mL ST solution (8.5 g L−1 NaCl, 1 mL L−1 Tween 80) was added to each well. A cotton swab was used to gently release spores from the agar. Spores were quantified using a haemocytometer.
Media recipes are based on minimal medium (MM), which contains 12.0 g L−1 NaNO3, 3.04 g L−1 KH2PO4, 1.04 g L−1, KCl, 1.04 g L−1, MgSO4.7H2O, and 1 mL L−1 Hutner's trace element solution (S. H. Hutner, L. Provasoli, A. Schatz, C. P. Haskins, Proc. Am. Phil. Soc. 1950, 94, 152-170; the entire disclosure of which is hereby incorporated by reference.). To create GMM and PPMNI, 3 g L−1 of d-glucose or cleaned PP digest were added to MM, respectively. Culture media were adjusted to pH 8.0 with 5.5 M KOH. For solid cultures, 15 g L−1 agar was added to the media. MM and GMM can be used as controls for this experiment.
While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention.
[1] U.S. Environmental Protection Agency, (EPA), “Advancing Sustainable Materials Management: 2018 Fact Sheet” (Publication 530-F-20-009, EPA, 2020; epa.gov/sites/default/files/2021-01/documents/2018_ff_fact_sheet_dec_2020_fnl_ 508.pdf).
[2] D. E. MacArthur, Beyond plastic waste. Science. 358, 843-843 (2017). 15 doi:10.1126/science.aao6749
[3] R. Geyer, J. R. Jamback, K. L. Law, Production, use, and fate of all plastics ever made. Sci.Adv. 3, e1700782 (2017). doi:10.1126/sciadv.1700782
[4] M. Eriksen, L. C. Lebreton, H. S. Carson, M. Thiel, C. J. Moore, J. C. Borerro, F. Galgani, P. G. Ryan, J. Reisser, Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic20 Pieces Weighing over 250,000 Tons Afloat at Sea. PloS one. 9, e111913 (2014). doi:10.1371/journal.pone.0111913
[5] A. A. de Souza Machado, W. Kloas, C. Zarfl, S. Hempel, M. C. Rillig, Microplastics as an emerging threat to terrestrial ecosystems. Global Change Biol. 24, 1405-1416 (2018). doi:10.1111/gcb.14020
[6] L. R. Melby, Nitric Acid Oxidation of High-Density Polyethylene. Organic Chemical Aspects. Macromolecules. 5 11, 50-56 (1978). doi:10.1021/ma60061a010
[7] A. Albertsson, S. Karlsson, Environment-adaptable polymers. Polym. Degrad. Stab. 41, 345-349 (1993). doi:10.1016/0141-3910(93)90082-T
[8] A. Pifer, A. Sen, Chemical Recycling of Plastics to Useful Organic Compounds by Oxidative Degradation. Angew. Chem. Int. Ed. 37, 3306-3308 (1998). doi:10.1002/(SICI)1521-3773 (19981217)37:23<3306::AID-ANIE3306>3.0.00,2-B
[9] X. Jia, C. Qin, T. Friedberger, Z. Guan, Z. Huang, Efficient and selective degradation of polyethylenes into liquid fuels and waxes under mild conditions. Sci. Adv. 2, e1501591 (2016). doi:10.1126/sciadv.150159
[10] G. Celik, R. M. Kennedy, R. A. Hackler, M. Ferrandon, A. Tennakoon, S. Patnaik, A. M. LaPointe, S. C. Ammal, A. Heyden, F. A. Perras, M. Pruski, S. L. Scott, K. R. Poeppelmeier, A. D. Sadow, M. Delferro, Upcycling Single-Use Polyethylene into High-Quality Liquid Products. ACS Cent. Sci. 5, 1795-1803 (2019). doi:10.1021/acscentsci.9b00722
[11] F. Kawi, T. Kawabata, M. Oda, Current state and perspectives related to the polyethylene terephthalate hydrolases available for biorecycling. ACS Sustain. Chem. Eng. 8, 8894-8908 (2020). doi:10.1021/acssuschemeng.0c01638
[12] C. M. Carr, D. J. Clarke, A. Dobson, Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives. Front. Microbiol. 11, 571265 (2020). doi:10.3389/fmicb.2020.571265
[13] J. Ru, Y. Huo, Y. Yang, Microbial degradation and valorization of plastic wastes. Front. Microbiol. 11, 442 (2020). doi:10.3389/fmicb.2020.00442
[14] V. Meyer, M. R. Andersen, A. A. Brakhage, G. H. Braus, M. X. Caddick, T. C. Cairns, R. P. de Vries, T. Haarmann, K. Hansen, C. Hertz-Fowler, S. Krappmann, U. H. Mortensen, M. A. Pellalva, A. F. J. Ram, R. M. Head, Fung. Biol. Biotech. 2016, 3, 6.
[15] J. W. Bok, D. Hoffmeister, L. A. Maggio-Hall, R. Murillo, J. D. Glasner, N. P. Keller, Chem. & Biol. 2006, 13, 31-37.
[16] V. Côté, G. Kos, R. Mortazavi, P. A. Ariya, Sci. Total Env. 2008, 390, 530-537.
[17] D. Y. Kim, Y. H. Rhee, Appl. Microbiol. Biotechnol. 2003, 61, 300-308.
[18] Y. Ishii, K. Nakayama, M. Takeno, S. Sakaguchi, T. Iwahama, Y. Nishiyama, J. Org. Chem. 1995, 60, 3934-3935.
[19] H. Gershon, L. Shanks, Can. J. Microbiol. 1976, 8, 1198-1201.
[20] M. T. Zumsteinarno, A. Schintlmeister, T. F. Nelson, R. Baumgartner, D. Woebken, M. Wagner, H. P. Kohler, K. Mcneill, M. Sander, Sci. Adv. 2018, 4, eaas9024.
[21] Y. M. Chiang, E. Szewczyk, A. D. Davidson, N. Keller, B. R. Oakley, C. C. Wang, J. Am. Chem. Soc. 2009, 131, 2965-2970.
[22] S. R. Paranjape, Y. M. Chiang, J. F. Sanchez, R. Entwistle, C. C. C. Wang, B. R. Oakley, T. C. Gamblin, Planta Med. 2014, 80, 77-85.
[43] K. Kaur, X. Wu, J. K. Fields, D. K. Johnson, L. Lan, M. Pratt, A. D. Somoza, C. C. C. Wang, J. Karanicolas, B. R. Oakley, L. Xu, R. N. De Guzman, PLoS One 2017, 12, e0175471.
[24] L. Lan, J. Liu, M. Xing, A. R. Smith, J. Wang, X. Wu, C. Appelman, K. Li, A. Roy, R. Gowthaman, J. Karanicolas, A. D. Somoza, C. C. C. Wang, Y. Miao, R. De Guzman, B. R. Oakley, K. L. Neufeld, L. Xu, Cancers 2020, 12, 2221.
[25] E. Szewczyk, T. Nayak, C. E. Oakley, H. Edgerton, Y. Xiong, N. Taheri-Talesh, S. A. Osmani, B. R. Oakley, Nat. Protoc. 2006, 1, 3111-3120.
[26] E. H. Creaser, R. L. Porter, K. A. Britt, J. A. Pateman, C. H. Doy, Biochem. J. 1985, 225, 449-454.
[27] P. J. Punt, M. A. Dingemanse, B. J. Jacobs-Meij sing, P. H. Pouwels, C. A. van den Hondel, Gene 1988, 69, 49-57.
[28] B. Felenbok, D. Sequeval, M. Mathieu, S. Sibley, D. I. Gwynne, R. W. Davies, Gene 1988, 73, 385-396.
[29] Y. M. Chiang, T. S. Lin, S. L. Chang, G. Ahn, C. C. C. Wang, ACS Synth. Biol. 2021, 10, 173-182.
[30] S. M. Poulsen, M. Karlsson, L. B. Johansson, B. Vester, Mol. Microbio. 2001, 41, 1091-1099.
[31] E. M. Gause, M. A. Buck, M. G. Douglas, J. Biol. Chem. 1981, 256, 557-559.
[32] H. R. Park, I. J. Ryoo, S. J. Choo, J. H. Hwang, J. Y. Kim, M. R. Cha, K. Shin-Ya, I. D. Yoo, Toxicology 2007, 229, 253-261.
[33] J. Sun, H. S. Alper, J. Ind. Microbiol. Biotechnol. 2015, 42, 423-436.
[34] A. Li, N. Pfelzer, R. Zuijderwijk, P. J. Punt, BMC Biotechnol. 2012, 12, 57.
[35] A. Li, N. van Luijk, M. ter Beek, M. Caspers, P. Punt, M. van der Werf, Fung. Gen. Biol. 2011, 48, 6.
[36] S. Krull, A. Hevekerl, A. Kuenz, U. PruBe, App. Microb. Biotech. 2017, 101, 4063-4072.
[37] Minisci, F., Punta, C., & Recupero, F. (2006). Mechanisms of the aerobic oxidations catalyzed by N-hydroxyderivatives. Journal of Molecular Catalysis A: Chemical, 251(1-2), 129-149. https://doi.org/10.1016/j.molcata.2006.02.011
[38] Rabot, C., Chen, Y., Bijlani, S., Chiang, Y.-M., Oakley, C. E., Oakley, B. R., Williams, T. J., & Wang, C. C. C. (2022). Conversion of Polyethylenes into Fungal Secondary Metabolites. Angewandte Chemie International Edition, anie.202214609. https://doi.org/10.1002/anie.202214609
[39] Boschloo, J. G., Moonen, E., van Gorcom, R. F. M., Hermes, H. F. M., & Bos, C. J. (1991). Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function. Current Genetics, 19 (4), 261-264. https://doi.org/10.1007/BF0035505244
[40] Borodina, I., Kenny, L. C., McCarthy, C. M., Parama-sivan, K., Pretorius, E., Roberts, T. J., van der Hoek, S. A., & Kell, D. B. (2020). The biology of ergothioneine, an antioxidant nutraceutical. Nutrition Research Re-views, 33 (2), 190-217.
[41] Cheah, I. K., & Halliwell, B. (2012). Ergothioneine; an-tioxidant potential, physiological function and role in disease. Biochimica et Biophysica Acta (BBA)—Molecu-lar Basis of Disease, 1822 (5), 784-793. https://doi.org/10.1016/j.bbadis.2011.09.01746
[42] Cheah, I. K., & Halliwell, B. (2021). Ergothioneine, re-cent developments. Redox Biology, 42, 101868. https://doi.org/10.1016/j.redox.2021.101868
[43] Seebeck, F. P. (2010). In Vitro Reconstitution of Myco-bacterial Ergothioneine Biosynthesis. Journal of the American Chemical Society, 132 (19), 6632-6633. https://doi.org/10.1021/ja101721e50
[44] Chiang, Y.-M., Szewczyk, E., Davidson, A. D., Keller, N., Oakley, B. R., & Wang, C. C. C. (2009). A Gene Cluster Containing Two Fungal Polyketide Synthases Encodes the Biosynthetic Pathway for a Polyketide, Asperfuranone, in Aspergillus nidulans. Journal of the American Chemical Society, 131(8), 2965-2970. https://doi.org/10.1021/ja8088185
[45] Yoshimi, A., Sano, M., Inaba, A., Kokubun, Y., Fujioka, T., Mizutani, O., Hagiwara, D., Fujikawa, T., Nishimura, M., Yano, S., Kasahara, S., Shimizu, K., Yamaguchi, M., Kawakami, K., & Abe, K. (2013). Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus. PLoS ONE, 8 (1), e54893. https://doi.org/10.1371/journal.pone.0054893
[46] Miyazawa, K., Yoshimi, A., Zhang, S., Sano, M., Naka-yama, M., Gomi, K., & Abe, K. (2016). Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase. Bioscience, Biotechnology, and Biochemistry, 80 (9), 1853-1863. https://doi.org/10.1080/09168451.2016.1209968
[47] Kavanagh, F., Hervey, A., & Robbins, W. J. (1951). Antibiotic Substances From Basidiomycetes. Proceedings of the National Academy of Sciences, 37 (9), 570-574. https://doi.org/10.1073/pnas.37.9.570
[48] Novak, R., & Shlaes, D. M. (2010). The pleuromutilin antibiotics: A new class for human use. Current Opinion in Investigational Drugs (London, England: 2000), 11 (2), 182-191.
[49] Poulsen, S. M., Karlsson, M., Johansson, L. B., & Vest-er, B. (2008). The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome. Molecular Microbiology, 41 (5), 1091-1099. https://doi.org/10.1046/j.1365-2958.2001.02595.x
[50] FDA approves new antibiotic to treat community-acquired bacterial pneumonia. (2019, Aug. 19).
[51] Alberti, F., Khairudin, K., Venegas, E. R., Davies, J. A., Hayes, P. M., Willis, C. L., Bailey, A. M., & Foster, G. D. (2017). Heterologous expression reveals the biosynthesis of the antibiotic pleuromutilin and generates bioactive semi-synthetic derivatives. Nature Communications, 8 (1), 1831. https://doi.org/10.1038/s41467-017-01659-1
[52] Chiang, Y.-M., Lin, T.-S., Chang, S.-L., Ahn, G., & Wang, C. C. C. (2021). An Aspergillus nidulans Plat-form for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products. ACS Synthetic Biology, 10 (1), 173-182. https://doi.org/10.1021/acssynbio.0c00536
[53] Rabot, C., Chen, Y., Bijlani, S., Chiang, Y.-M., Oakley, C. E., Oakley, B. R., Williams, T. J., & Wang, C. C. C. (2022). Conversion of Polyethylenes into Fungal Sec-ondary Metabolites. Angewandte Chemie International Edition, anie.202214609. https://doi.org/10.1002/anie.202214609
[54] Ritter, S. K. What's That Stuff? Motor Oil. Chem. Eng. News 2006, 84, https://cen.acs.org/articles/84/i11/Motor-Oil.html
[55] (a) Abdalla, N.; Fehrenback, H. LCA for Regeneration of Waste Oil to Base Oil. Institut fur Energie and Umweltforschung Heidelberg (IFEU), Heidelberg, 14 Apr. 2018. https://www.ifeu.de/fileadmin/uploads/ifeu-GEIR-LCA-regeneration-waste-oil_final-version-2018corr.pdf (b) Rerefining Used Lubrication and Industrial Oils' Effects on Virgin Crude Oil Refining. California Department of Resources Recycling and Recovery. Report to the Legislature, 6 Feb. 2015. https://www2.calrecycle.ca.gov/Publications/Details/1518 (c) Geyer, R.; Kuczenski, B.; Henderson, A.; Zink, T. Life Cycle Assessment of Used Oil Management in California. California Department of Resources Recycling and Recovery. Publication # DRRR-2013-1465. 29 Jul. 2013.
[56] Gershon, H.; Shanks, L. Antifungal Properties of a,w-Alkanedicarboxylic Acids and their Dimethyl Esters. Can. J. Microbiol. 1976, 8, 1198-1201.
This application claims the benefit of U.S. provisional application Ser. No. 63/416,085 filed Oct. 14, 2022, the disclosure of which is hereby incorporated in its entirety by reference herein.
This invention was made with government support under Grant No. CHE-1856395 awarded by the National Science Foundation (NSF) and Contract No. DE-AC05-76RL01830 awarded by the U.S. Department of Energy (DOE). The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63416085 | Oct 2022 | US |
