Applications and biosynthesis of cyanobacterial compounds

Abstract

Disclosed herein are methods of biosynthesizing scytonemin, including monomeric and dimeric forms and analogs thereof. Additionally, disclosed herein are compositions comprising scytonemin or an analog thereof, such as compositions suitable for topical application to the skin of a subject. Also disclosed herein are methods of using scytonemin compositions in both clinical and cosmetic applications relating to treatment of the skin.

Description

APPLICATIONS AND BIOSYNTHESIS OF CYANOBACTERIAL COMPOUNDSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 515,510, filed on July 25, 2023, and entitled “APPLICATIONS AND BIOSYNTHESIS OF CYANOB ACTERIAL COMPOUNDS,” the entire disclosure of which is expressly incorporated by reference herein.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML copy, created on July 16, 2024 is named "SunecoST26" and is 55,645 bytes in size.FIELD OF THE INVENTION

[0003] The disclosure relates to methods of use of the monomer of scytonemin in practice and biosynthesis of the UV absorbing compound scytonemin. The disclosure further relates to compositions comprising scytonemin, and methods of using the same.BACKGROUND

[0004] Skin cancers represent the most common malignancies in the United States, and ultraviolet (UV) radiation and sunburn are clear risk factors for development of skin cancer. The vast majority of dermatologists recommend use of sunscreens to prevent sunburn, skin cancer, and photoaging. However, recent evidence suggests that chemical sunscreens such as avobenzone, oxybenzone, and octocrylene may present significant health risks including endocrinologic disruption, malignancy, and end-organ toxicity. In response to these findings, many European regulatory agencies have limited use of these products.

[0005] The environmental effects of these compounds are equally concerning. Coral reefs and the ecosystems they support are undergoing a mass extinction event via coral bleaching. Chemical sunscreens have been shown to deposit in coral from bathers and degrade into photo-toxins. This discovery prompted the state of Hawaii to ban chemical sunscreen ingredients. Chemical UV absorbers are also used in a broad variety of industrial and consumer applications, raising concern for human and environmental health. Mineral sunscreens including titanium and zinc oxide are the only current alternative to chemical products. In concentrations needed for appropriate photoprotection, mineral sunscreens create a thick, mask-like white sheen that is cosmetically unacceptable, especially for consumers with skin-of-color. For these reasons, new compounds are needed on the market that are safe, environmentally friendly, and cosmetically elegant.

[0006] Many natural compounds are synthesized by phytoplankton for photoprotection. These compounds absorb UV radiation across a wide spectrum and show great promise as adjuvant agents in sunscreens and other cosmeceuticals. Additionally, some of these compounds exhibit potent anti-inflammatory, anti-proliferative, antioxidant, or skin brightening properties. However, they are only produced in small quantities by these organisms, which presents challenges to production and industrial use. One such natural product of emerging interest is scytonemin.

[0007] Scytonemin is dimeric indole alkaloid synthesized by cyanobacteria ("blue-green algae" or "phytoplankton") for photoprotection. It absorbs ultraviolet (UV) radiation across a broad spectrum with peaks at 300 nm (UVB) and 376 nm (UVA). Despite its powerful photoprotective properties, several challenges limit its inclusion in commercial products. In one example, the complexity of its biosynthesis is one barrier to efficient production, specifically achieving dimerization of monomer of scytonemins. Under natural conditions, the dimerization occurs in the complex redox environment of the cyanobacteria's periplasm. Accordingly, there exists a need forimproved biosynthesis strategies not only for scytonemin but also for precursors and derivatives thereof. Additionally, there is a need for compositions including scytonemin, such as cosmetic formulations and photoprotective products. Aspects of the invention disclosed herein address these needs.INCORPORATION BY REFERENCE

[0008] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually, and as if each is fully set forth herein. However, where such reference is made, and whether to patents, publications, non-patent literature, or other sources of information, it is for the general purpose of providing context for discussing features of the invention. Accordingly, unless specifically stated otherwise, the reference is not to be construed as an admission that the document or underlying information, in any jurisdiction, is prior art, or forms part of the common general knowledge in the art.SUMMARY OF THE INVENTION

[0009] This invention relates to advanced methods and compositions for the biosynthesis of scytonemin and its analogues, including scytonemin monomers. Scytonemin, a natural indole alkaloid dimer produced by cyanobacteria is a potent absorber of ultraviolet (UV) radiation in UVA and UVB and blue light spectra. These properties are particularly useful in preventing skin damage, photoaging, and other deleterious effects associated with UV exposure.

[0010] The invention provides novel methods for the efficient biosynthesis of scytonemin and its derivatives, which overcome the limitations posed by the complex natural biosynthesis pathways. These methods involve the use of heterologous expression platforms to express scytonemin biosynthesis enzymes and transporter proteins in microbial host organisms or use ofcell-free or immobilized enzyme systems. Specific embodiments include the expression of scytonemin biosynthesis proteins, such as ScyA, ScyB, ScyC, ScyD, ScyE, and ScyF derived from Nostoc punctiforme in E. coli, facilitating the commercial production of scytonemin monomer or dimer and / or and dimerization of scytonemin monomers.

[0011] Additionally, the invention includes methods of use of scytonemin, scytonemin monomer, and analogues including novel compositions comprising scytonemin, scytonemin monomers, or analogues thereof, along with various enhancements such as zinc oxide, antioxidants like vitamin C and E, and skin-beneficial agents, like niacinamide. These compositions are formulated for applications in skincare, dermatology, or to improve the appearance of skin. Compositions using scytonemin offer benefits such as enhanced UV protection, antiinflammatory, antioxidant, and improved cosmetic formulation without the undesirable whitening effect common with traditional mineral sunscreens. Compositions using scytonemin monomer offer skin brightening, anti-inflammatory, and antioxidant effects.

[0012] The disclosed methods and compositions not only address the need for scaling and formulation of novel UV protective and skincare products but also the other uses of scytonemin, the scytonemin monomer, and their analogues including as skin brightening and antioxidant agents.BRIEF DESCRIPTION OF THE FIGURES

[0013] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description serve to explain the principles of the disclosure.

[0014] FIG 1. Chemical structure of scytonemin (middle) in theoretical reduced (left) and oxidized (right) states.

[0015] FIG 2. Chemical structure of the 18C compound synthesized prior to scytonemin dimerization in vivo, referred to herein as the scytonemin monomer.

[0016] FIG 3. Schematic showing an exemplary mixed modality biosynthesis involving dimerization of a scytonemin monomer by a cell-free system, which results in separation of scytonemin from the aqueous solution due to hydrophobicity.

[0017] FIG 4. Schematic showing an exemplary cell-free method of biosynthesizing scytonemin intermediates followed by dimerization of scytonemin monomer (red signage over the ScyE protein refers to removal of signalling or trafficking sequences from the enzymes).

[0018] FIG 5. Schematic showing exemplary components for engineering E. coli to biosynthesize scytonemin, including plasmids encoding scytonemin synthesis enzymes and the Ebo transporter.

[0019] FIG 6. List of exemplary plasmids for use in engineering an E. coli heterologous expression host to produce scytonemin, including pScyABCE, pEBO, pScyABCE:Asig, pScy:Ebo, pScyABCEF, and pScyABCEF:Asig.

[0020] FIG 7. Schematic showing exemplary application of scytonemin or derivatives thereof (“Natural UV-filters”) and a reduced amount of other UV-modulating compounds (ZnO and TiOi) in a sunscreen composition.

[0021] FIG 8. Graph showing the inhibition of tyrosinase of scytonemin monomer compared to hydroquinone, kojic acid, and arbutin.

[0022] FIG 9. Table showing the anti-oxidant, free-radical scavenging activity of scytonemin monomer in lipophilic medium.

[0023] FIG 10. Photograph of various preparations of sun protection formulations incorporating scytonemin as a UVA / UVB absorption booster.DETAILED DESCRIPTION

[0024] Biosynthesized by cyanobacteria, the natural product scytoncmin has been recognized for its UV-absorbing, anti-inflammatory, and antioxidant properties. Scytonemin is a brownish- yellow, lipid-soluble pigment that is excreted and accumulates in the extracellular matrix of cells exposed to UV-A radiation (315-400 nm) (Garcia-Pichel and Castenholz, J. Phycol. 1991 ;27: 395- 409; Garcia-Pichel et al., Photochem. Photobiol. 1992;56: 17-23; Rastogi et al., Chemosphere 2013;93 1874-1878; Rastogi and Incharoensakdi, FEMS Microbiol. Ecol. 2014;87: 244-256). Structurally unique among natural products, scytonemin is a homodimeric indole-alkaloid, with a molecular mass of 544 g / mol, composed of two heterocyclic units (cyclopentyl[b]indole) that are symmetrically connected through a carbon-carbon bond. FIG. 1 shows the chemical structure of scytonemin along with theoretical reduction and oxidation states of the compound. The complex ring structures allow a strong absorption in the UV-A-violet-blue range (325-425 nm), with a maximum of 384 nm in acetone and around 370 nm in vivo (Garcia-Pichel and Castenholz, J. Phycol. 1991;27: 395-409; Proteau et al., Experientia 1993;49 825-829).

[0025] Enzymes involved in biosynthesis of scytonemin include ScyA, ScyB, ScyC, ScyD, ScyE, and ScyF. Orthologous sequences of cyanobacterial proteins, and the respective genes coding for them, may be retrieved at the NCBI (National Center for Biotechnology Information) database, e.g., after Protein-Protein Basic Local Alignment Search Tool (BLASTp) analysis against the respective scytonemin biosynthesis proteins from N. punctiforme . It has been speculated that scytonemin biosynthesis is compartmentalized in the cell, with the monomersynthesis and initial condensation occurring in the cytoplasm, and the later reactions, such as dimerization, taking place in the periplasm, prior to pigment excretion. Enzymes encoded by scyD, scyE, and scyF may be periplasmic proteins, and would logically be involved in the late steps of the scytonemin biosynthetic pathway (Soule et al., BMC Genomics 2009; 10:336; Gao and Garcia- Pichel, Nat. Rev. Microbiol. 2011;9 791-802). The ebo gene cluster, which is associated with the scytonemin operon of cyanobacteria, has been implicated in the excretion of scytonemin intermediates from the cytoplasm into the periplasm during biosynthesis. See, e.g., Klicki et al. mBio. 2018 Nov 27;9(6):e02266-18 and Klicki et al, 2021, Doctoral Dissertation, Arizona State University.

[0026] ScyE is part of the beta-propeller clan, a functionally heterogeneous protein family, and is predicted to be periplasmic due to the presence of putative signal peptides (Soule et al., J. Bacteriol. 2007; 189 4465-4472; Soule et al., BMC Genomics 2009; 10:336; Ferreira & Garcia- Pichel, Front Microbiol. 2016; 7: 735). Although the role of ScyE in scytonemin biosynthesis is not fully understood, the protein may be involved in oxidative dimerization of the scytonemin monomer in the periplasm, although such activity has never been demonstrated. See, e.g., Ferreira & Garcia-Pichel, Front Microbiol. 2016; 7: 735 and Klicki et al, 2021, Doctoral Dissertation, Arizona State University. Whereas dimerization of the scytonemin monomer has been previously achieved by chemical modification, now described are methods of dimerizing the scytonemin monomer using biosynthesis enzymes ScyD, ScyE, ScyF, or protein products of the Ebo gene cluster, which provide the exemplary advantage of efficient dimerization at various scales of production.

[0027] Herein, the terms “analog” and “analogs” refer to any compound that shares similarities in structure or function with another compound, such as scytonemin. The term is intended toencompass chemical compounds derived from scytonemin, such as derivatives, and includes precursors and chemical modifications thereof. Exemplary scytonemin analogs are disclosed in, e.g., He et al., Front Microbiol. 2022 Jul 13; 13:912621 , Vamali & Edwards, Philos Trans A Math Phys Eng Sci. 2014 Dec 13;372(2030):20140197, Grant & Louda, Organic Geochemistry, 2013;65:29-36, W01996006607A1, US20220331218A1, WG2020128112A1, and an exemplary precursor is provided in FIG. 2.

[0028] Herein, “therapeutically effective amount” or “therapeutically effective dose” of a composition, e.g., a composition comprising scytonemin or an analog thereof, refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts will typically depend upon the EC50 and safety profile of the specific agent being administered.

[0029] Biosynthesis

[0030] In some aspects, provided herein are methods of biosynthesizing scytonemin, such as monomeric and scytonemin. Scytonemin biosynthesis may be accomplished using cell-based methods, cell-free techniques, or a combination thereof. The synthesis of scytonemin, including precursors and analogs thereof, may be achieved using a heterologous expression involving a microbe, such as a bacteria cell or a yeast cell. Specifically, heterologous expression systems can be used to produce the scytonemin monomer and precursors thereof. Methods of biosynthesizing scytonemin are described in, e.g., E. coli (Malla and Sommer, Green Chem., 2014, 16, 3255-3265) and Nostoc punctiforme (Ferreira & Garcia-Pichel, Front Microbiol. 2016; 7: 735). Herein, methods of dimerizing scytonemin monomers involve organic synthesis techniques or enzymatic modification, such as cell-free or cell-based enzymatic modification.

[0031] FIG. 3 and FIG. 4 shown exemplary schemes of scytonemin biosynthesis. As shown in FIG. 3, such methods can involve a heterologous expression system. Exemplary biosynthesismethods involving, e.g., ScyE and other scytonemin biosynthesis enzymes, are represented by both FIG. 3 and FIG. 4, where ScyM represents scytonemin monomers.

[0032] In some embodiments, disclosed methods comprise producing scytonemin with use of a microbe and a heterologous expression system. In some embodiments, disclosed methods comprise use of thin-layer raceway ponds, closed culture, or photobioreactors in the synthesis of scytonemin and analogs thereof.

[0033] In some examples, scytonemin biosynthesis involves genetically manipulating organisms which naturally produce scytonemin to optimize the expression of the same and analogs thereof. Natural producers express constituents of the scytonemin operon and include but are not limited to the following genera: Nostoc, e.g., N. commune, N. sphaeroides, N. calciole, or N. punctiforme, Synechocystis, Spirulina, Arthrospira, e.g., Arthrospira platensis, Lyngbya, Scylonema. Tolypothrix, or Gloebacter.

[0034] Metabolic engineering of Nostoc spp. including but not limited to N. punctiforme to target genes regulating expression of DNA products such as RNA and proteins with influence on expression of scytonemin, its precursors, or its analogs. Exemplary techniques involved in disclosed methods include but are not limited to any manipulation of the two-factor regulatory system, e.g., insertion of promoters, inhibitors, or any other regulatory apparatus which may materially affect the expression of scytonemin or an analog thereof.

[0035] In some embodiments, the scytonemin monomer is produced by a microbial host organism, such as a bacteria cell or a yeast cell. In some embodiments, the scytonemin monomer is produced by E. coli, N. punctiforme, S. cerevisiae, B. subtilis, or S epidermis.

[0036] In some embodiments, disclosed methods comprise dimerizing the scytonemin monomer. In some embodiments, the scytonemin monomer is not produced by E. coli, N.punctiforme, S. cerevisiae, B. subtilis, or S epidermis. Tn some embodiments, the scytonemin monomer is produced by chemical synthesis.

[0037] In some embodiments, disclosed methods comprise transforming a microbial host organism with any one or more of pScyABCE, pEBO, pScyABCE: Asig, pScy:Ebo, pScyABCEF, pScyABCEF:Asig.

[0038] The aforementioned plasmids include the ScyABCE genes with a modified signaling peptide sequence. In some examples, modification of the N-terminal peptide signaling sequence results in cytoplasmic expression of the protein product.

[0039] In some examples, a cell-free system is used to synthesize of scytonemin, such as the scytonemin monomer and analogs thereof. This may include expression and stabilization of enzymes involved in scytonemin biosynthesis. Such enzymes include but are not limited to tyrosine aminotransferase, ScyA, ScyB, ScyC, ScyE, and any combination thereof. This necessarily includes all steps involved in the process of building a cell-free system for the purposes of constructing cyanobacteria metabolites such as stabilization and folding of protein products, provision of reaction energy, and optimization of process dynamics. In some examples, the cell- free approach may be combined with any of the disclosed methods to optimize synthesis strategies. The scytonemin monomer can be synthesized using ScyA, Scy B, and ScyC. However, for dimerization, ScyA, ScyB, ScyC, ScyE, optionally ScyF and the Ebo operon are necessary.

[0040] In some embodiments, disclosed methods comprise dimerizing the scytonemin monomer. In some embodiments, disclosed methods comprise contacting the scytonemin monomer or a precursor thereof with a recombinant scytonemin biosynthesis enzyme, such as ScyD, ScyE, or ScyF. In some embodiments, disclosed methods comprise contacting the scytonemin monomer or a precursor thereof with an ebo gene cluster protein product, such as EboA, Ebo B, Ebo C, Ebo D, or Ebo F. In some embodiments, disclosed methods comprise contacting the scytonemin monomer or a precursor thereof with recombinant scytonemin biosynthesis protein ScyD, ScyE, ScyF, Ebo A, Ebo B, Ebo C, Ebo D, Ebo F, or a combination thereof. In some embodiments, disclosed methods comprise contacting the scytonemin monomer or a precursor thereof with recombinant ScyE enzyme.[00411 In some embodiments, the recombinant scytonemin biosynthesis protein is immobilized on a surface, such as a solid matrix. In some embodiments, the solid surface is a microplate, a membrane, a chip, a slide, a bead, a magnetic bead, a nanoparticle, or a microfluidic device. In some embodiments ScyD, ScyE, ScyF, Ebo A, Ebo B, Ebo C, Ebo D, Ebo F, or a combination thereof is immobilized on a surface.

[0042] In some embodiments, disclosed methods comprise dimerizing the scytonemin monomer with a recombinant scytonemin biosynthesis enzyme, e.g., ScyD, ScyE, and ScyF, wherein the recombinant enzyme is not expressed by a cell, such as a cell-free recombinant scytonemin biosynthesis enzyme. The cellular components of a model organism such as E coli are extracted and placed in preparation with DNA templates, amino acids, nucleoside triphosphates, energy systems, and supplements. The enzyme RNA is transcribed and translated in vitro and then purified. In some embodiments, the cell-free recombinant scytonemin biosynthesis enzyme is immobilized on a surface, such as a solid matrix. In some embodiments, the solid surface is a microplate, a membrane, a chip, a slide, a bead, a magnetic bead, a nanoparticle, or a microfluidic device. In some embodiments, the recombinant scytonemin biosynthesis enzyme is ScyD, ScyE, ScyF, or a combination thereof.

[0043] Under natural conditions, such as in wild-type cyanobacteria, the dimerization of scytonemin monomers occurs via an unknown mechanism, possibly in the periplasm of thecyanobacteria. Whereas Malla et al. attempted to design plasmids encoding ScyE, the enzyme is notably absent from SDS PAGE evidence of scytonemin enzymes produced by their engineered E. coli strains. See Figure SI of Malla and Sommer, Green Chem.. 2014, 16, 3255-3265, which shows detection of scyA, scyB, and scyC, but not scyE. In some examples, described herein are methods of combining cell-free expression of recombinant scytonemin biosynthesis enzymes, including ScyE, and heterologous expression of the ebo channel in E. coli or other model organisms to produce dimerized scytonemin or analogs thereof.

[0044] In some examples, disclosed methods comprise modifying scytonemin or an analog thereof to alter the compound’s light-absorbing characteristics. In some embodiments, such modification comprises glycosylation, methylation, oxidation, or a combination thereof.

[0045] In some embodiments, disclosed methods comprise separation of scytonemin from a mixture, such as an aqueous solution. Routine laboratory, production, or manufacturing techniques may be used to separate mixture components by polarity from an aqueous solution. Exemplary techniques involve use of separatory funnelling or chromatography of non-polar products, e.g., as represented in FIG. 3. In some embodiments, disclosed methods comprise separating scytonemin or an analog thereof from a mixture by crystallization.

[0046] Compositions

[0047] In some aspects, provided herein are compositions comprising scytonemin or an analog thereof. In some embodiments, disclosed compositions are suitable for topical application to a subject in need thereof, preferably a human. Disclosed compositions have UVA, UVB, and blue light absorption properties, which may be determined according to methods available to one of skill in the art, e.g, spectrophotometric analysis, photostability, sun protection factor testing (SPF), critical wavelength testing, and persistent pigment darkening testing.

[0048] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof and a microbe, such as the microbe that produced scytonemin or the analog thereof. In some embodiments, the microbe naturally biosynthesizes scytonemin or an analog thereof. In some embodiments, the microbe is a heterologous expression host engineered to produce scytonemin or an analog thereof. In some embodiments, disclosed compositions comprise purified scytonemin or an analog thereof, such as a cell-free preparation.In some embodiments, disclosed compositions comprise the scytonemin monomer, scytonemin, or a combination thereof. In some embodiments, disclosed compositions consists of the scytonemin monomer or an analog therof. In some embodiments, the disclosed composition consists of scytonemin or an analog thereof. In some embodiments, the scytonemin monomer or an analog thereof is crystallized and / or purified. In some embodiments, scytonemin or an analog thereof is crystallized and / or purified. Such a composition may be used to ameliorate, treat, or improve appearance of a skin condition, or improve tolerability of another formulation. Scytonemin, scytonemin monomers, or their analogues function as free-radical scavengers, antioxidants, or any iteration of an antiinflammatory agent.

[0049] In some embodiments, scytonemin, scytonemin monomer, or their analogues can be solubilized in a lipophilic solvent. Exemplary lipophilic solvents include C 12-15 alkyl benzoate, C16-17 alkyl benzoate, behenyl benzoate, isopropyl benzoate, isobutyl benzoate, dipropylene glycol dibenzoate, or similar lipophilic solvent. Such lipophilic solvents may act as skin-conditioning agents and emollients.

[0050] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof in an % amount (w / w) of 0.01% to 5%, 0.01% to 10%, 0.01% to 15%, 0.01% to 20%,0.01 % to 25%, 0.01 % to 30%, 0.01 % to 35%, 0.01 % to 40%, 0.01 % to 45%, 0.01 % to 50%, 0.01 % to 55%, 0.01% to 60%, 0.01% to 65%, 0.01% to 70%, 0.01% to 75%, 0.01% to 80%, 0.01% to 85%, 0.01% to 90%, 0.01% to 95%, or 0.01% to 100%,

[0051] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof in an % amount (w / w) of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0052] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof in combination with a chemical sunscreen and / or a physical sunscreen. Disclosed compositions may comprise scytonemin, precursors, and derivatives thereof in combination with active ingredients that serve to enhance sun protection factor (SPF) or UVA protection factor or high-energy visible light (e.g. blue light, >380 nm). An exemplary advantage of a composition comprising scytonemin in combination with a chemical and / or physical sunscreen is that the UV- absorbing properties of scytonemin allow for use of relatively low concentrations of the chemical and / or physical sunscreen to achieve an SPF relative to a comparator formulation composed primarily of the chemical and / or physical sunscreen. FIG. 7 shows additional exemplary advantages of disclosed compositions, including SPF enhancement, such as synergistic increase in SPF protection, a cosmetically superior formulation, anti-inflammatory and anti-aging effects, decreased erythema upon sun exposure, and free-radical scavenging effects.

[0053] In some examples, disclosed compositions comprise scytonemin having a modified oxidation state, wherein the compound exhibits improved physical properties, including but not limited to light absorption, solubility, emulsification, texture, appearance, or interaction with other compounds. Disclosed compositions may comprise scytonemin in a partially or fully oxidized state, achieved, e.g., by chemical modification, such that the light-absorbing properties are enhanced relative to scytonemin in vivo.

[0054] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof, in combination with a chemical sunscreen. In some embodiments, the chemical sunscreen is or comprises avobenzone, octinoxate, octisalate, homosalate, octocrylene, oxybenzone, terephthalyidene dicamphor sulfonic acid, drometrizole trisiloxane, bis-ethylhexyloxyphenol methoxyphenyl triazine, or a combination thereof. In some embodiments, disclosed compositions do not comprise a chemical sunscreen. In some embodiments, disclosed compositions do not comprise avobenzone, octinoxate, octisalate, homosalate, octocrylene, oxybenzone, terephthalyidene dicamphor sulfonic acid, drometrizole trisiloxane, bis-ethylhexyloxyphenol methoxyphenyl triazine, or a combination thereof.

[0055] In some embodiments, disclosed compositions comprise scytonemin or an analog thereof in combination with a physical or chemical (“organic”) sunscreen. In some embodiments, the physical sunscreen is or comprises zinc oxide, titanium dioxide, iron oxide, or a combination thereof. Indicative of favorable properties, such as formulation homogeneity, a surprising interaction was observed upon formulating scytonemin with zinc, titanium, and other metals, specifically the metals appeared to be complexed with scytonemin, including analogs and derivatives thereof.

[0056] Additional inorganic compounds may be included in disclosed compositions to enhance formulation properties, e.g., spreadability, stability, and appearance, e.g., whiteness or tint. In some embodiments, disclosed compositions further comprise talc, silica, alumina, magnesium stearate, mica, sodium chloride, aluminum hydroxide, or a combination thereof.

[0057] In some embodiments, disclosed compositions further comprise a pigment. In some embodiments, the pigment is a carotenoid, a cartenoprotein, a xanthopyll, or a porphyrin- based / heme-porphyrin based pigment. Inclusion of pigments or color additives in disclosed compositions can be used to modify the wavelength absorption of a composition. Such pigments or light-absorbing or reflecting small molecules may be derived from bacteria, fungi, or plants. See, e.g., US20050129630A1, W02000024369A1, and WO2022250557A1.

[0058] In some embodiments, disclosed compositions further comprise a modulator of melanin production, alternatively referred to as a melanin production modulator. Representative modulators of melanin production include tyrosinase inhibitors, MITF regulators, melanogenesis signaling pathway modulators, receptor-targeting agents, melanosome transfer inhibitors, antioxidants, hormonal modulators, melanocyte- specific cytotoxic agents, precursor regulators, and glutaminergic and adrenergic pathway modulators. Exemplary melanogenesis signaling pathway modulators regulate the activity of any of the cAMP pathway, PKC pathway, MEK / ERK pathway, and Wnt pathway. Exemplary receptor-targeting agents modulate the activity of any of melanocortin- 1 receptor (MC1R), tyrosine kinase receptor (c-KIT), frizzled receptor, and endothelin B receptor (ETBR).

[0059] In some embodiments, disclosed compositions further comprise mycosporine, gadusols, oxo-mycosporines, imino-mycosporines, mycosporine-like amino acids (MAAs), melanines, flavonoids, betalanines, tetrahydrobiopterin, phenylpropanoids, polyphenols, tannins,pycnogenol, tyrosinases, alpha hydroxy acids (AHAs), polysaccharides, glycosaminoglycans (GAGs), mucopolysaccharides, vitamin E, polymers, collagen, keratin, elastin, linoleic acid, laminin, tretinoin, tazarotene, sargaquinoic acid, sargachromenol, fucoxanthin, retinoid, antiinflammatory cytokines (as 11-2), cortisone, tacrolimus, ciclosporin, resveratrol, gallocatechol, gallocatechin, epigallocatechin gallate, ferulic acid, ubiquinone, lycopene, astaxanthin, retinoid, vitamin A and derivatives thereof, beta-carotene, vitamin D and derivatives thereof, cortisone, tacrolimus, ciclosporin, photolyase, endonuclease, glycosylase, or a combination thereof.

[0060] In some embodiments, disclosed compositions further comprise kojic acid, tranexamic acid, niacinamide, hydroquinone, arbutin, azelaic acid, ascorbic acid, tocopherol, cysteamine, rescorinol, retinoids, thiamidol, fungal-derived pigment modulators, derivatives thereof, or a combination thereof. Such compounds may be useful for skin-brightening, management of hyperpigmentation, pathologic dyspigmentation, and other cosmetic indications recognized by those skilled in the art.

[0061] In some embodiments, disclosed compositions further comprise a penetration enhancer or a surfactant. In some embodiments, the penetration enhancer is or comprises dimethyl sulfoxide (DMSO), oleic acid, propylene glycol, or a combination thereof. In some embodiments, disclosed compositions comprise a liposomal preparation for delivery of hydrophobic materials into the epidermis.

[0062] In some examples, the scytonemin monomer functions as an antioxidant or free radical scavenger.

[0063] In some examples, scytonemin monomer, dimer, analog, precursor, or derivative thereof may be formulated to enhance solubility and penetration, such as penetration into the epidermis. In some embodiments, disclosed compositions comprise scytonemin formulated in aliposome, a microemulsion, a nanoemulsion, a solid lipid nanoparticle, a nano structured lipid carrier, a cyclodextrin, or an ethosome.

[0064] In some embodiments, disclosed compositions further comprise a dispersing agent. In some embodiment, the dispersing agent comprises a polymer, a surfactant, a lipid, a silica-based particle, a cyclodextrin, a protein, or a combination thereof.

[0065] In some embodiments, a scaffolding molecule such as a protein, protein fragment, or polysaccharide is used to enhance the dispersion of scytonemin.

[0066] In some embodiments, scytonemin is used to enhance, support, or otherwise affect dispersion of another ingredient in formulation including but not limited to zinc oxide or titanium dioxide.

[0067] In some embodiments, scytonemin interacts with metal ions such as iron, zinc, or titanium to form a complex or non-covalent, covalent, ionic, or other interaction.

[0068] In some embodiments, a protein product such as water stress proteins such as but not limited to the protein product of wspa in Nostoc commune or other peptide or molecule is used in formulation with scytonemin for the purpose of providing non-covalent interactions. These interactions may enhance the stability, photostability, shelf-life, or performance of scytonemin.

[0069] In some embodiments, structural molecules derived from the cyanobacterial extracellular glycan sheath are used for stabilization of scytonemin in formulation.

[0070] In some embodiments, a quenching agent is used in conjunction with scytonemin for the purposes of enhancing stability, especially when exposed to light. Such agents may include but are not limited to antioxidants such as Vitamin C vitamin E, resveratrol, green tea extracts or molecules derived from them such as EGCG (Epigallocatechin Gallate), ubiquinone, carotenoids,polyphenolic or metabolite extracts from plants, fungi, or bacteria, or other known ultraviolet light filters. These may be used individually or in any combination.

[0071] In other embodiments, scytonemin or the scytonemin monomer is encapsulated or micro-encapsulated to enhance skin penetration or provide stability. Any method of encapsulation may be used as known by those skilled in the art.

[0072] In some embodiments, scytonemin is used as a quenching agent for other ultraviolet or energy absorbing ingredients, such as but not limited to avobenzone, oxybenzone, bemotrizinol, octinoxiate, octisalate, homosalate, octocrylene, salicylate derivatives including butyloctyl salicylate, ecamsule, dioxybenzone, aminobenzoic acid (PABA), phenylbenzimidazole, sulisobenzone, trolamine, and / or cinoxate .

[0073] In some embodiments, disclosed compositions of formulations have a sun protection factor (SPF) of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more, or any integer or derivative therein. In some embodiments, disclosed compositions may be formulated as a lotion, a spray, a cream, a stick, a gel, an ointment, a suspension, or a powder.

[0074] Method of Use

[0075] In some aspects, provided herein are methods of using scytonemin or an analog thereof, such disclosed scytonemin compositions, in clinical and cosmetic applications relating to the skin. In addition to their photoprotective effects, disclosed compositions may be used to treat a skin condition. In some embodiments, disclosed methods comprise administering compositions comprising scytonemin to a subject in need thereof. In some embodiments, disclosed compositions are administered to the subject at least once per day. In some embodiments, disclosed methods comprise topically administering scytonemin to a subject in need thereof, such as a mammal, preferably a human.

[0076] Scytonemin Monomer as an Active Ingredient

[0077] In some examples, scytonemin monomer is used as an active ingredient in formulations, especially to improve a subject’s skin appearance including evenness of the subject’s skin tone or to brighten the skin or as an anti-oxidant or anti-aging agent. These applications differ substantially from previously proposed methods of use, which involve “skin-whitening,” also known as “skin bleaching.” Such terms specifically refer to the intent to induce a generalized hypopigmentation, or intent to reduce pigmentation below basal expression for a subject, rather than targeting of localized dyspigmentation, hyperpigmentation, or skin brightening.

[0078] In some embodiments, disclosed methods comprise administering a disclosed composition to a subject suffering from a pigmentation disorder. In some embodiments, the pigmentation disorder is post-inflammatory hyperpigmentation, melasma, solar lentigines, ephelides, cafe au lait macules, erythema dyschromicum perstans, vitiligo, poikiloderma of civatte, photodamage, photoaging, skin changes that have been associated with aging, or hyperpigmentation associated with Addison’s disease.

[0079] In some embodiments, disclosed methods comprise administering scytonemin monomer, scytonemin, or a combination thereof to a subject in need thereof. In some embodiments, disclosed methods comprise administering the scytonemin monomer or an analog therof to a subject in need thereof. In some embodiments, disclosed methods comprise administering scytonemin or an analog thereof to a subject in need thereof. In some embodiments, the scytonemin monomer or an analog thereof for administration to a subject is crystallized and / or purified. In some embodiments, scytonemin or an analog thereof for administration to a subject is crystallized and / or purified.

[0080] Aims of treating pigmentation disorders, such as desired therapeutic results, include, e.g., lightening or reducing the appearance of dark spots or patches, preventing additional pigmentation, improving skin brightness, reducing photodamage or the appearance of photodamage, or improving skin appearance related to aging. Disclosed compositions may additionally ameliorate inflammation, itchiness, or dryness, which may accompany hyperpigmentation .

[0081] In some embodiments, the scytonemin monomer is modified and an analogue is created for the purposes of reducing reactivity and increasing tolerability.

[0082] Methods of determining treatment efficacy resulting from use of disclosed compositions, such as topical administration of a therapeutically effective amount to the skin of a subject in need thereof, are within the knowledge of one of skill in the art. Exemplary methods of determining treatment efficacy, which are dependent upon the condition being treated, include visual inspection, patient-reported outcome measures, clinical trials, clinician-evaluated outcomes including use of a validated scale such as melasma area and severity index (MASI), point counting serial image index, skin hyperpigmentation index (SHI) skin tone measurement, Wood’s lamp examination, dermoscopy, photography including digital, a digital platform including but not limited to Mexameter, DermaSpectrometer, Chroma Meter, VISIA, Antera 3D, and ImageJ along with all other software platforms developed with the intention of evaluating the efficacy or outcome of a product that could be construed to be useful for skin, biopsy, reflectance confocal microscopy, skin swabs or scrapings, and patch testing.

[0083] Co-administration

[0084] In some embodiments, disclosed methods comprise co-administering a disclosed composition with kojic acid, tranexamic acid, niacinamide, hydroquinone, arbutin, azelaic acid,ascorbic acid, tocopherol, cysteamine, rescorinol, a retinoid, thiamidol, fungal -derived pigment modulators, derivatives thereof, or a combination thereof to a subject in need thereof.

[0085] In some embodiments disclosed methods comprise co-administering a disclosed composition with mycosporine, gadusols, oxo-mycosporines, imino-mycosporines, mycosporine- like amino acids (MAAs), melanines, flavonoids, betalanines, tetrahydrobiopterin, phenylpropanoids, polyphenols, tannins, pycnogenol, tyrosinases, alpha hydroxy acids (AHAs), polysaccharides, glycosaminoglycans (GAGs), mucopolysaccharides, vitamin E, polymers, collagen, keratin, elastin, linoleic acid, laminin, tretinoin, tazarotene, sargaquinoic acid, sargachromenol, fucoxanthin, retinoid, anti-inflammatory cytokines (as 11-2), cortisone, tacrolimus, ciclosporin, resveratrol, gallocatechol, gallocatechin, epigallocatechin gallate, ferulic acid, ubiquinone, lycopene, astaxanthin, retinoid, vitamin A and derivatives thereof, beta-carotene, vitamin D and derivatives thereof, cortisone, tacrolimus, ciclosporin, photolyase, endonuclease, glycosylase, or a combination thereof.EXAMPLES

[0086] Example 1: Production of Scytonemin

[0087] E. coli is engineered to produce scytonemin monomers, as previously described by Malla and Sommer, Green Chem.. 2014, 16, 3255-3265. Briefly, E. coli BL21(DE3) carrying plasmids necessary for scytonemin monomer synthesis, including pCDF-ScyACD and pACYC- ScyB, is grown in standard culture media supplemented with tryptophan and tyrosine. A crude extract of the centrifuged cell pellet is obtained. The presence of scytonemin monomer is detected by LCMS. The solution of the crude extract is then placed in a cell-free lysate comprising any one or more of recombinant ScyD, ScyE, ScyF, and scytonemin monomer (ScyM) (FIG. 3). Alternatively, the scytonemin monomer contacts any one or more of recombinant ScyD, ScyE,ScyF immobilized on an enzyme matrix (FIG. 4). Dimerized scytonemin is harvested from the solution by separatory funneling.

[0088] Example 2: Production of Scytonemin from Exemplary Heterologous Expression Host E. coll

[0089] A heterologous E. coli model expressing enzymes necessary for synthesis of the scytonemin monomer is designed. FIG. 5 shows an exemplary method of engineering a microbe, such as E. coli, to produce scytonemin, involving plasmids encoding scytonemin synthesis enzymes and the ebo transporter, along with supplementation of tyrosine and tryptophan. FIG. 6 shows exemplary plasmids which may be used to facilitate biosynthesis of scytonemin. The scytonemin monomer is crystallized. The crystallized scytonemin monomer is reconstituted in an aqueous solution accommodating the oxidation / reduction environment of a scytonemin biosynthesis protein, e.g., ScyD, ScyE, or ScyF. Immobilized scytonemin biosynthesis proteins are fixed to a surface. Flow channels direct the solution containing scytonemin monomer solution over the immobilized enzymes, which are supplied with magnesium ion co-factor and a recycling system for flavin adenine dinucleotide. Dimerized scytonemin is produced.

[0090] Example 3: Production of Scytonemin Monomer From a Gram Positive Heterologous Expression Host

[0091] A heterologous Gram positive bacterial model, such as B subtilis or .S' epidermidis, is engineered to express enzymes necessary for synthesis of the scytonemin monomer. A plasmid is engineered expressing the scytonemin operon using a high efficiency plasmid origin such as pBAV!K-T5, e.g., as described in Bryksin & Matsumura, Biotechniques . 2010 Jun;48(6):463-5, which encodes scytonemin biosynthesis enzymes and ebo transporter proteins, along withsupplementation of tyrosine and tryptophan. FIG. 6 shows exemplary plasmids which may be used to facilitate biosynthesis of the scytonemin monomer.

[0092] The scytonemin monomer is crystallized. The crystallized scytonemin monomer is reconstituted in an aqueous solution accommodating the oxidation / reduction environment of one or more dimerizing scytonemin biosynthesis enzymes, e.g., including but not limited to ScyE. Immobilized scytonemin biosynthesis proteins are fixed to a surface. Flow channels direct the solution containing scytonemin monomer solution over the immobilized enzymes, which are supplied with magnesium ion co-factor and a recycling system for flavin adenine dinucleotide. Contact between the scytonemin monomer and recombinant scytonemin biosynthesis enzymes produces scytonemin.

[0093] Example 4: Purification of Scytonemin

[0094] E. coli BL21(DE3) is transformed using standard protocols to express plasmids with genes coding for N. punctiforme proteins Scy A, Scy B, Scy C, Scy E, and eboABCDF. The cell culture is centrifuged and a pellet is obtained. The biomass is shaken with 1:1 chloroforrmhexane then centrifuged. The supernatant is removed and the procedure is repeated. The biomass is washed with ethylacetate:methanol and centrifuged. The process is again repeated until the supernatant is clear. The remaining sediment is washed with hexane. The presence of highly pure scytonemin is confirmed using HPLC.

[0095] Example 5: Sunscreen Formulation

[0096] A composition comprising zinc oxide 5% (w / w), scytonemin 0.5% (w / w), and antioxidants in a caprylic / capric triglyceride base is suitable for topical application and use as a sunscreen. Exemplary antioxidants include vitamin C, vitamin E, niacinamide, and ferulic acid.The photoprotective effects of the composition are assessed using in vitro and in vivo measures,e.g., the Broad Spectrum Test, which provides an in vitro measurement of a sunscreen drug product’s ability to protect against both UVA and UVB radiation, and the SPF Test, which provides an in vivo clinical measurement of a sunscreen drug product’ s ability to protect against sunburn, which is primarily mediated by UVB. Provided the UV-absorbing activity of scytonemin or analogs thereof, reduced amounts of physical sunscreen, such as ZnO or TiO2 may be necessary for formulation to achieve a certain SPF, as represented by FIG. 7.

[0097] Example 6: Lotion Formulation

[0098] A composition comprising scytonemin monomer (0.5% w / w) and kojic acid (2.5% w / w) is formulated in a vehicle suitable to produce lotion. Alternatively, a composition comprising scytonemin monomer (<1% w / w) and niacinamide (5% w / w) is formulated in a vehicle suitable to produce lotion. Compositions are suitable for topical application and may be used in the treatment or to improve appearance of certain skin conditions, including, e.g., melasma and post- inflammatory hyperpigmentation.

[0099] Example 7: Administration & Treatment Efficacy

[0100] A disclosed composition, such as a composition comprising scytonemin or an analog thereof, is applied to the skin of a subject in need thereof. The subject suffers from a pigmentation disorder, such as melasma or post-inflammatory hyperpigmentation. The composition is administered to the subject at least once per day. Treatment efficacy is determined using a combination of observer- and subject-reported measures, e.g., a combination of visual inspection, skin tone measurement, and a patient-reported measure, such as a questionnaire or survey.

[0101] Example 8: Tyrosinase Activity

[0102] A method for evaluating the inhibition of tyrosinase enzyme activity by test compounds is disclosed, comprising the following steps: preparing an assay buffer consisting of PBS with 1%Triton X- 100, and extracting tyrosinase from normal human epidermal melanocytes in the assay buffer. A stock solution of the substrate dihydroxy-phenyl-alanine L3,4 (L-DOPA) is prepared at 10 mM in the assay buffer. The test compound or the reference compound, kojic acid (at a concentration of 0.071 mg / ml for the first assay or at concentrations of 0.125, 0.25, 0.5, and 1 mM for the second assay), is pre-incubated with the enzyme for 10 minutes on ice. Following this preincubation, L-DOPA is added to achieve a final concentration of 2 mM, and the mixture is incubated at 37 °C for 1 hour. The enzymatic activity is then evaluated by measuring the optical density at 540 nm using a microplate reader, with additional optical density measurements taken without substrate addition to detect potential interference from the test or reference compounds at each concentration.

[0103] FIG. 8 shows the comparative tyrosinase activity of a control sample, hydroquinone, arbutin, kojic acid, and HC-01.

[0104] Example 9: Determination of Antioxidant and Chelation Properties

[0105] Antioxidant and chelating properties of test compounds were determined by electrochemical analysis in lipophilic medium.

[0106] A method for performing an electrochemical test using electrochemistry-based technology, comprising the following steps: the sample is first diluted in an electrolyte solution and then deposited onto a sensor. An electric potential variation (E in V / Reference Electrode) is applied using the device, and the evolution of the corresponding electric current (i in A) is recorded. After a basic signal treatment, the potential -current curve can be interpreted as follows: a peak with a positive amplitude indicates an oxidation process, such as corrosion or metallic dissolution, where a reductant is converted to an oxidant with the release of electrons (Reductor —> Oxidant + electron(s)); a peak with a negative amplitude indicates a reduction process, such asthe formation of metallic salts, where an oxidant accepts electrons to become a reductant (Oxidant + electron(s) — Reductor).

[0107] FIG. 9 shows the antioxidant activity of Euk 134 and 1415686-24-6 in oxidizing conditions (5mM H2O2).

[0108] Example 10: Comparative Textural Properties of Sunscreen Formulations

[0109] A method for incorporating scytonemin into the oil stage of an oil-in-water emulsion for cosmetic formulations is disclosed, comprising the steps of preparing the oil phase by combining oil-soluble ingredients, including scytonemin, under appropriate mixing conditions. Scytonemin is added to the oil phase at concentrations of 0.2% and 0.5% weight by weight, where 0.2 grams of scytonemin is added per 100 grams of the oil phase for the 0.2% concentration, and 0.5 grams per 100 grams for the 0.5% concentration. The oil phase containing scytonemin is heated and mixed to ensure even distribution and complete dissolution of scytonemin. Separately, the water phase ingredients are prepared and heated. The oil phase is then gradually added to the water phase under high-shear mixing to form a homogenous oil-in-water emulsion. The emulsion is cooled to room temperature with continuous stirring.

[0110] FIG. 10 shows a comparison of sunscreen formulations. The textural properties of market sunscreen formulations (SPF 40, 44, and 50 from left to right) were contrasted with formulations containing 0.2% and 0.5% scytonemin. A slight green tinge is evident at 0.5% resulting from increasing the concentration of oxidized scytonemin.EQUIVALENTS AND SCOPE

[0111] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of thepresent invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0112] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0113] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0114] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specificallyset forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0115] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranged can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0116] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of the ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the ail. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5% or up to 1% of a given value. Alternatively, the term can mean within an order of magnitude, for example within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0117] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from any one or more claims.

[0118] Sequence Table

Claims

CLAIMSI claimClaim 1. A composition comprising scytonemin or an analog thereof, zinc oxide, caprylic / capric triglyceride, and an antioxidant.Claim 2. The composition of claim 1, wherein the antioxidant is selected from the group consisting of vitamin C, vitamin E, niacinamide, ferulic acid, and a derivative thereof.Claim 3. A composition comprising scytonemin, monomer of scytonemin, or an analog thereof with or without a modulator of melanin production.Claim 4. The composition of claim 3, wherein scytonemin or the analog thereof is monomeric or dimeric.Claim 5. The composition of claim 3, wherein monomer of scytonemin, scytonemin, or the analog thereof is purified.Claim 6. The composition of claim 3, wherein monomer of scytonemin is or the analog thereof is crystallized.Claim 7. A method of treating a subject in need thereof comprising topically administering the composition of claim 1 to the subject.Claim 8. The method of claim 7, wherein the subject has a pigmentation disorder.Claim 9. The method of claim 7, wherein the pigmentation disorder comprises melasma or post inflammatory hyperpigmentation or “age spots” (solar lentigo), aging, photoaging, photodamage, or a combination thereof.Claim 10. The method of claim 7, wherein the subject is human.Claim 11. A method for biosynthesizing scytonemin monomer or an analog thereof comprising expressing a scytonemin biosynthesis enzyme and an ebo transporter protein in a microbial host organism.Claim 12. The method of claim 11, wherein the recombinant scytonemin biosynthesis protein is selected from the group consisting of recombinant ScyA, ScyB, ScyC, Scy D, ScyE, and Scy F.Claim 13. The method of claim 11, wherein the ebo transporter protein is selected from the group consisting of Ebo A, Ebo B, Ebo C, Ebo D, and Ebo F.Claim 14. The method of claim 1 1 , further comprising purifying the scytonemin monomer.Claim 15. The method of claim 1 1 , further comprising dimerizing the scytonemin monomer by contacting the scytonemin monomer with recombinant ScyD, ScyE, ScyF, or a combination thereof.Claim 16. The method of claim 11, wherein the microbial host is E. coli, N. punctiforme, S. cerevisiae, B. subtilis, or S epidermis.Claim 17. A method for dimerizing the scytonemin monomer comprising associating multiple monomers of scytonemin using a recombinant scytonemin biosynthesis protein.Claim 18. The method of claim 17, wherein the recombinant scytonemin biosynthesis protein is immobilized on a matrix.Claim 19. The method of claim 17, wherein the recombinant scytonemin biosynthesis protein is selected from the group consisting of recombinant Scy D, ScyE, and Scy F.Claim 20. The method of claim 17, comprising solubilizing scytonemin in a lipophilic solvent.

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