I. RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/291,718, filed on December 20, 2021. The content of U.S. Provisional Application No. 63/291,718 is hereby incorporated by reference in its entirety.

II. SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled DEBU-01 l-01WO-37396-70-Sequence-Listing_ST26, created on December 19, 2022 and having a size of 24 KB. The content of the sequence listing is incorporated herein its entirety.

III. FIELD OF THE INVENTION

The present invention features a method of producing astaxanthin and other derivatives by enzyme-modification of beta-carotene and its derivatives. In particular, the present invention features a cell-free production method.

IV. BACKGROUND OF THE INVENTION

Carotenoids are naturally occurring yellow, orange, and red pigments. There are over 1100 known carotenoids found in plants, algae, bacteria, and fungi. Carotenoids are the source of color for many fruits and vegetables and other animals and plants such as salmon, canaries, shrimp, egg yolks, marigolds, and some autumn leaves. Carotenoids can be further categorized into xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons and contain no oxygen).

Astaxanthin is a keto-carotenoid that widely exists in nature and is the end of carotenoid synthesis in vivo. Additionally, astaxanthin itself is bright red and responsible for the red color of Crustacea, molluscs and salmons, which cannot synthesize astaxanthin de novo. In the body, astaxanthin has various excellent biological properties such as preventing UV radiation, protecting retinas and central nervous systems, preventing cardiovascular disease and enhancing immunity of an organism.

V. SUMMARY OF THE INVENTION

However, manufacturing of carotenoids via chemical synthesis or in cells suffers from problems that limit the commercial viability of high-value chemical production. Chemical synthesis requires extensive, elaborate, expensive, toxic, and inefficient multi-step chemical reactions to produce natural products that often are too complex to make in the laboratory. Manufacturing processes involving bio-foundries (use of the whole cell) suffer from product toxicity, carbon flux redirection, diffusion problems through cell walls, and toxic byproduct generation.

It is an objective of the present invention to provide a cell-free method that allows for the production of astaxanthin and other derivatives of beta-carotene, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

In the cell-free systems described herein, the critical components of the cell, namely cofactors and enzymes, are used in a chemical reaction without cellular components that can directly or indirectly inhibit the desired biochemical reaction. The same enzymes found in plants and other organisms may be created in vivo (typically through protein overexpression in hosts such as bacteria), isolated via chromatography, and then added into a bioreactor with a substrate (starting material). The enzymes transform the substrate in the same way that occurs in the original organism without the organism’s complexity. Additionally, the biochemical reaction may be enhanced by the addition of co-solvents, detergents, or both, which would not be tolerated by, or simply would not work in a whole cell-based manufacturing method. In this way, natural products can be created without the plant, cell, or chemical synthesis.

In some embodiments, the present invention features a cell-free method of converting beta-carotene to a keto-carotenoid or a hydroxylated carotenoid. In some embodiments, the method comprises adding to a mixture comprising a suitable solvent (1) beta-carotene and (2) a betacarotene hydroxylase enzyme (CrtZ), a beta-carotene ketolase enzyme (CrtW) or a combination thereof, to form a reaction mixture. In some embodiments, the method comprises removing the supernatant from the aforementioned reaction mixture. In some embodiments, the method comprises isolating the hydroxylated carotenoid or the keto-carotenoid.

In other embodiments, the present invention may also feature a cell-free method of converting beta-carotene to astaxanthin or an intermediate product. In some embodiments, the method comprises adding to a mixture comprising a suitable solvent (1) beta-carotene and (2) a beta-carotene hydroxylase enzyme (CrtZ), a beta-carotene ketolase enzyme (CrtW) or a combination thereof, to form a reaction mixture. In some embodiments, the method comprises removing the supernatant from the aforementioned reaction mixture. In some embodiments, the method comprises isolating astaxanthin or the intermediate product. In some embodiments, the intermediate product is beta-cryptoxanthin, zeaxanthin, echinenone, canthaxanthin, 3’- hydroxyechinenone, phoenicoxanthin, 3-hydoxyechinenone, adonixanthin or a combination thereof.

A unique and inventive technical feature of the present invention is the use of a cell-free system for the production of astaxanthin and other intermediate products from P-carotene. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for higher reaction concentrations of one or more of starting materials (substrates, e.g., P-carotene and P-cryptoxanthin), reagents, and/or enzymes, resulting in higher concentrations of final products (e.g., astaxanthin). Additionally, the present invention eliminates the complications of cell walls, thereby eliminating a significant barrier to product and substrate diffusion. Furthermore, the present invention eliminates competition for carbon flux, which limits the efficiency of cell-based synthesis methods, and thus greatly reduces byproduct formation. Also, because there is no cell, the present invention is not vulnerable to degradation by cell death due to the formation of toxic compounds. In addition, the present methods enhance the ability to use various solvents, such as organic solvents, to permit higher concentrations of solutes (e.g., substrates and intermediates) without worrying about killing the cell.

Furthermore, the previously known techniques teach away from the present invention. For example, prior methods of producing astaxanthin require synthesis of astaxanthin in a living organism (e.g., microalgae (e.g., Haematococcus pluvialis) or plants from the family Ranunculacea) and then extraction of the astaxanthin.

Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

VI. BRIEF DESCRIPTION OF THE THE DRAWING(S)

The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which: FIG. 1 shows Carotenoid Biosynthesis from the KEGG pathway

FIG. 2 shows a simplified carotenoid biosynthesis pathway showing multiple substrates/products for many enzymes.

FIG. 3 shows a detailed beta-carotene to astaxanthin pathway. Two enzymes (CrtZ and CrtW) are required and produce up to 8 intermediates (as shown in FIG. 4).

FIG. 4 shows that the present invention can produce 8 intermediate carotenoids in the conversion of beta-carotene to astaxanthin.

FIG. 5 shows a sample time course experiment for some of the major products produced by the present invention.

VII. DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, and/or methods are disclosed and described, it is to be understood that, unless specified, this invention is not limited to specific synthetic methods or to specific compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Additionally, although embodiments of the disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not provide all the features and benefits described herein. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and/or uses and obvious modifications and equivalents thereof. Moreover, while a number of variations have been shown and described in varying detail, other modifications, which are within the scope of the present disclosure, will be readily apparent to those of skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form varying modes of the present disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described herein.

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

As used herein, “reaction solution” may refer to all components necessary for enzymebased chemical transformation. This is typically, but not limited to, buffering agent, salts, cofactor, and substrate (starting material).

As used herein, “reaction mixture” may refer to all components from the “reaction solution” plus the enzyme(s) and/or products from the reaction. In some embodiments, the “reaction mixture” may refer to just the reaction solution without any enzymes or reaction products.

As used herein, “reaction mixture” may refer to all components from the “reaction solution” plus the enzyme(s) and/or products (mono- or di-hydroxylated carotenoids or ketocarotenoids) of the reaction. In some embodiments, “reaction solution” and “reaction mixture” may be used interchangeably

As used herein, “buffering agents” may refer to chemicals added to water-based solutions that resist changes in pH by the action of acid-base conjugate components.

As used herein, “supernatant” may refer to the soluble liquid fraction of a sample.

As used herein, “batch reactions” may refer to a chemical or biochemical reaction performed in a closed system such as a fermenter or typical reaction flask.

As used herein, “cofactors” may refer to a non-protein chemical compound that may bind to a protein and assist with a biological chemical reaction. Non-limiting examples of cofactors may include but are not limited to NADPH and NADH.

As used herein “lysate” may refer to a fluid containing the products of cellular lysis. Lysis is the action of breaking down the cellular membrane and can be achieved by multiple mechanisms including but not limited to enzymatic, osmotic, or mechanical mechanisms.

Percent molar ratio (i.e., “% (molar ratio)”) is the molar ratio of product produced by a reaction relative to substrate used in the reaction.

As used herein, a “suitable solvent” refers to a substance that dissolves a solute (e.g., betacarotene) and results in a solution. In other embodiments, a suitable solvent is a substance which is compatible with the enzymes used herein (e.g., a solvent in which an enzyme’s activity is retained).

As used herein, “purity” refers to the absence of impurity or contaminants in a composition. In some embodiments, purity of a composition described herein may be measured using techniques well known in the art.

Referring now to FIGs. 1-5, the present invention features a cell free method of producing astaxanthin as well as other intermediates from P-carotene and its derivatives.

The present invention may feature a cell-free method of converting beta-carotene to a keto-carotenoid or a hydroxylated carotenoid. In some embodiments, the method comprises adding to a mixture comprising a suitable solvent (1) beta-carotene and (2) a beta-carotene hydroxylase enzyme (CrtZ), a beta-carotene ketolase enzyme (CrtW) or a combination thereof, to form a reaction mixture. In some embodiments, the method comprises removing the supernatant from the aforementioned reaction mixture. In some embodiments, the method comprises isolating the hydroxylated carotenoid or the keto-carotenoid.

Non-limiting examples of hydroxylated carotenoids include but are not limited to betacryptoxanthin or zeaxanthin. Non-limiting examples of keto-carotenoids include but are not limited to echinenone, canthaxanthin, or astaxanthin.

In some embodiments, the beta-carotene is converted to beta-cryptoxanthin, zeaxanthin, echinenone, canthaxanthin, 3 ’-hydroxy echinenone, phoenicoxanthin, 3-hy doxy echinenone, adonixanthin, astaxanthin or a combination thereof.

In some embodiments, the isolated hydroxylated carotenoid has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

In other embodiments, the isolated hydroxylated carotenoid has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%. In some embodiments, the isolated keto-carotenoid has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

In other embodiments, the isolated keto-carotenoid has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

The present invention may also feature a cell-free method of converting beta-carotene to astaxanthin or an intermediate product. In some embodiments, the method comprises adding to a mixture comprising a suitable solvent (1) beta-carotene and (2) a beta-carotene hydroxylase enzyme (CrtZ), a beta-carotene ketolase enzyme (CrtW) or a combination thereof, to form a reaction mixture. In some embodiments, the method comprises removing the supernatant from the aforementioned reaction mixture. In some embodiments, the method comprises isolating astaxanthin or the intermediate product.

In some embodiments, the intermediate product is beta-cryptoxanthin. In other embodiments, the intermediate product is zeaxanthin. In further embodiments, the intermediate product is selected from a group consisting of beta-cryptoxanthin, zeaxanthin, echinenone, canthaxanthin, 3 ’-hydroxy echinenone, phoenicoxanthin, 3 -Hy doxy echinenone, and adonixanthin.

The cell-free methods of preparing hydroxylated caretnoids, including beta-cryptoxanthin and zeaxahnthin are also disclosed in PCT/US2022/073874, which is incorporated herein by reference in its entirety.

In some embodiments, the isolated astaxanthin has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

In other embodiments, the isolated astaxanthin has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

In some embodiments, the purity of the isolated astaxanthin depends on how long the reaction as described herein runs (e.g., the longer the reaction runs the purer the astaxanthin isolated from the method becomes).

In some embodiments, the isolated astaxanthin is formulated into a liquid. In other embodiments, the isolated astaxanthin is formulated into an oil. In further embodiments, the isolated astaxanthin is formulated into a powder.

In some embodiments, the beta-carotene has a concentration in the reaction mixture of about 1.0 mM to about 30 mM. In other embodiments, the beta-carotene has a concentration in the reaction mixture of about 1.0 mM to about 30 mM, or about 1.0 mM to about 20 mM, or about 1.0 mM to about 10 mM, or about 10 mM to about 30 mM, or about 10 mM to about 20 mM, or about 20 mM to about 30 mM.

In some embodiments, the enzymes may be immobilized. In some embodiments, immobilized enzymes may be immobilized onto solid supports. Non-limiting examples of solid supports may include (but are not limited to) epoxy methacrylate, amino Ce methacrylate, or microporous polymethacrylate. In further embodiments, various surface chemistries may be used for linking the immobilized enzyme to a solid surface, including but not limited to covalent, adsorption, ionic, affinity, encapsulation, or entrapment. In other embodiments, the enzymes are non-immobilized. Either immobilized or non-immobilized enzymes may be employed in batch or continuous synthesis. For example, an immobilized enzyme on a solid support may be used in a cartridge through which a reaction mixture passes, whereby an immobilized enzyme may catalyze modification of substrate to produce the product at a high titer. Alternatively, a continuous method may comprise micro mixing of enzyme solution and substrate to produce the product at a high titer, while continuously removing product, removing (e.g., recovering) substrate, or both. In some embodiments removed (e.g., recovered) substrate may be recycled to increase process efficiency and overall yield.

In some embodiments, the beta-carotene hydroxylase enzyme (CrtZ) or the beta-carotene ketolase enzyme (CrtW) are non-immobilized. In other embodiments, the beta-carotene hydroxylase enzyme (CrtZ) or the beta-carotene ketolase enzyme (CrtW) are immobilized. In further embodiments, the beta-carotene hydroxylase enzyme (CrtZ) or the beta-carotene ketolase enzyme (CrtW) are within a continuous reactor system.

[0001] In some embodiments, the enzymes used in the methods described herein are wild type enzymes from various bacterial species such as late-stage enzymes in the Crt pathways of bacteria in the Pantoea and Brevundimonas genera. In other embodiments, the enzymes used in the methods described herein are variants of the wild type enzymes from various species.

In certain embodiments, described herein, the CrtW enzyme used is AB181388.1 and the CrtZ enzyme used is CRH37458.1. However, the present invention is not limited to the enzymes disclosed herein and may include other homologous enzymes comprising the same activity (i.e., the same hydroxylase and/or ketolase activity).

Table 1 shows non-limiting examples of Beta-carotene Hydroxylase Enzymes that may be used in accordance with methods described herein.

Table 2 shows non-limiting examples of Beta-carotene Ketolase Enzymes that may be used in accordance with methods described herein

In some embodiments, the ratio of CrtZ:CrtW enzymes varies. This ratio is important because CrtW may have a different efficiency as compared to CrtZ. In some embodiments, CrtW is much more efficient than CrtZ. In some embodiments, the ratio of CrtZ:CrtW is about 2:1, or about 2.5:1, or about 3:1. In other embodiments, the ratio of CrtZ:CrtW is about 20:1, or about 15:1, or about 10:1, or about 5:1, or about 2:1, or about 1:1, or about 1:2, about 1:5, or about 1:10, or about 1:15, or about 1:20. In some embodiments, the ratio of CrtZ:CrtW is about 20:1 to about 1:20, about 15:1 to about 1:20, about 10:1 to about 1:20, about 5:1 to about 1:20, about 2:1 to about 1:20, about 1:1 to about 1:20, about 1:2 to about 1:20, about 1:5 to about 1:20, or about 1:10 to about 1:20. In some embodiments, the ratio of CrtZ:CrtW is about 20:1 to about 1:15, about 15:1 to about 1:15, about 10:1 to about 1:15, about 5:1 to about 1:15, about 2:1 to about 1:15, about 1:1 to about 1:15, about 1:2 to about 1:15, about 1:5 to about 1:15, or about 1:10 to about 1:15. In some embodiments, the ratio of CrtZ: CrtW is about 20:1 to about 1:10, about 15: 1 to about 1:10, about 10:1 to about 1:10, or about 5:1 to about 1:10, about 2:1 to about 1:10, about 1:1 to about 1:10, about 1:2 to about 1:10, or about 1:5 to about 1:10. In some embodiments, the ratio of CrtZ:CrtW is about 20:1 to about 1:5, about 15:1 to about 1:5, about 10:1 to about 1:5, about 5:1 to about 1:5, about 2: 1 to about 1:5, about 1 : 1 to about 1 :5, or about 1:2 to about 1 :5. In some embodiments, the ratio of CrtZ:CrtW is about 20:1 to about 1:2, about 15:1 to about 1:2, about 10:1 to about 1:2, about 5 : 1 to about 1 :2, about 2: 1 to about 1 :2, or about 1 : 1 to about 1 :2. In some embodiments, the ratio of CrtZ:CrtW is about 20:1 to about 1:1, about 15:1 to about 1:1, about 10:1 to about 1:1, about 5 : 1 to about 1 : 1 , or about 2 : 1 to about 1 : 1. In some embodiments, any of the foregoing ratios of CrtZ:CrtW may be reversed (i. e. , the ratio of CrtW:CrtZ may be any of the ratios recited above for CrtZ: CrtW).

In other embodiments, the amount of CrtZ and CrtW enzymes are changed through modifying gene dosage or copy number to better balance the enzymes in the organisms. Without wishing to limit the present invention to any theories or mechanisms it is believed that the gene dosage and/or copy number of the CrtZ and CrtW enzyme genes may be modified in a variety of ways to balance these enzymes in the organism. Non-limiting examples include but are not limited to 1) modifying a single plasmid to include multiple copies of the same gene (i.e., CrtZ or CrtW), 2) integrating multiple copies of the same gene (i.e., CrtZ or CrtW) into the genome of the host organism, 3) changing to the DNA sequence preceding the gene on the plasmid or integrated into the host genome that are designed to increase or decrease the expression of each gene.

In some embodiments, the reaction condition may comprise 6% THF, 1% Tween-20, 50 pM Iron(ii), about 600 mg/L beta-carotene at pH 8 run at 16°C overnight. The longer the reaction runs the more Astaxanthin (and less intermediates) are present.

In some embodiments, the suitable solvent may comprise THF at 6%. Suitable solvents include, but are not limited to, e.g., tetrahydrofuran (THF), dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetonitrile (ACN), or mixtures or combinations of two or more thereof. In reference to % (v/v), “about” indicates ± 10% of the stated percentage (e.g., “about 0.5% (v/v)” indicates 0.5% (v/v) ± 0.05% (v/v) and “about 20% (v/v)” indicates 20% ± 2% (v/v)).

In preferred embodiments, the reaction mixture comprises about 6% THF. In some embodiments, the reaction mixture comprises about 1% THF, or about 2% THF, or about 3% THF, or about 4% THF, or about 5% THF, or about 6% THF, or about 7% THF, or about 8% THF, or about 9% THF, or about 10% THF. In preferred embodiments, the reaction mixture comprises 1% Tween-20. In some embodiments, the reaction mixture comprises about 0.2% Tween-20, or about 0.4% Tween-20, or about 0.6% Tween-20, or about 0.8% Tween-20, or about 1% Tween-20, or about 1.5% Tween-20, or about 2% Tween-20.

In preferred embodiments, the reaction mixture comprises 50 pM Iron(ii). In some embodiments, the reaction mixture comprises about 20 pM Iron(ii), or about 25 pM Iron(ii), or about 30 pM Iron(ii), or about 35 pM Iron(ii), or about 40 pM Iron(ii), or about 45 pM Iron(ii), or about 50 pM Iron(ii), or about 55 pM Iron(ii), or about 60 pM Iron(ii), or about 65 pM Iron(ii), or about 70 pM Iron(ii), or about 75 pM Iron(ii).

In preferred embodiments, the reaction mixture comprises 600 mg/L of beta-carotene (i.e., substrate/starting material). In some embodiments, the reaction mixture comprises about 300 mg/L, or about 350 mg/L, or about 400 mg/L, or about 450 mg/L or about 500 mg/L or about 550 mg/L or about 600 mg/L, or about 650 mg/L, or about 700 mg/L, or about 750 mg/L, or about 800 mg/L, or about 850 mg/L, or about 900 mg/L, or about 950 mg/L, or about 1000 mg/L, or about 1100 mg/L, or about 1200 mg/L, or about 1300 mg/L, or about 1400 mg/L, or about 1500 mg/Lof beta carotene (i. e. , substrate/starting material).

In preferred embodiments, the pH of the reaction is 8.0. In some embodiments, the pH of the reaction may range from about 7.0 to about 9.0. In some embodiments, the pH of the reaction may be about 8.5 (e.g., about 7.0 to about 9.0, about 7.5 to about 9.0, about 8.0 to about 9.0, about 8.2 to about 8.8, about 8.3 to about 8.7, about 8.4 to about 8.6). In some embodiments, the pH of the reaction may be in the range of 8.2 to 8.7 or 8.3 to 8.6. In each case of pH, “about” indicates ± 0.1 pH unit.

In some embodiments, the temperature of the reaction may range from about 10 °C to about 25 °C. In some embodiments, the temperature of the reaction may be about 15 °C, e.g., about 10 °C to about 20°C, about 12 °C to about 18 °C, or about 14 °C to about 16 °C. In each case of °C, “about” indicates ± 1 °C.

The reaction time may be varied to optimize yield or to balance yield against efficient use of resources. The reaction time may vary from 1 hour to 48 hours. In some embodiments, the time to run the reaction may range from about 5 hours to about 15 hours, e.g., about 5 hours to about 10 hours, about 5 hours to about 8 hours, about 5 hours to about 7 hours, or about 5 to about 6 hours. In each case of time, “about” indicates ± 0.5 hr. In some embodiments, if the reaction is stopped early all the peaks (i.e. , the intermediates) can be seen (FIG. 4), if the reaction is run to completion only astaxanthin is seen.

As used herein, the term “about” refers to plus or minus 10% of the referenced number.

Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met. Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.