COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This applicaton claims the benefit of U.S. Provisional Patent Application No. 63/161,162, filed 15 March 2021, which is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB [0002] The content of the ASCII text file of the sequence listing named “PT1069_ST25.txt” which is 24.1 kb in size created on March 9, 2022 and electronically submitted vis EFS-Web herewith the application is incorporated by reference in its entirety.

BACKGROUND

[0003] Demand for plant-based meat substitutes is increasing for a variety of reasons. Many consumers prefer meat substitute options that perform most similarly to animal meat, including wanting the color of the meat substitute to be comparable to animal meat color before and after cooking. Accordingly, there is a need for a pigment that can provide color to a meat substitute that is the same or similar to that of natural animal meat. A pigment derived from natural sources is particularly desirable.

SUMMARY

[0004] The present disclosure provides pigment compositions for a meat substitute comprising a red chromogenic protein (RCP) in an amount effective for increasing the red color of a meat substitute. The RCP can be a cnidarian RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. S SAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001. The RCP can comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one sequence selected from the group consisting of SEQ ID NOs:l-12 and comprises an XYG chromophore tripeptide. The RCP can have an absorbance spectrum maximum between 450 nm and 600 nm. When the pigment is heated at 80 °C for 20 minutes, the absorbance of light at 580 nm is at least 80% of the absorbance at 580 nm prior to heating or greater than 100% of the absorbance at 580 nm prior to heating. [0005] The disclosure further provides a meat substitute comprising a non-meat protein and an RCP. The RCP can be a cnidarian RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. S SAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001. The RCP can comprise a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one sequence selected from the group consisting of SEQ ID NOs:l-12 and comprises an XYG chromophore tripeptide. The RCP can have an absorbance spectrum maximum between 450 nm and 600 nm. The meat substitute can comprise 0.01% to 6%, 0.05% to 5%, 0.1% to 3%, or 0.5% to 2% by weight of the RCP or a pigment composition comprising the RCP.

[0006] The meat substitute can comprise a plant-based protein, a fungal-derived protein, an insect protein, and in vitro cultured animal cell, or combinations thereof. The non-meat protein can comprise a plant-based protein selected from the group consisting of pea protein, soy protein, corn protein, chickpea protein, and wheat protein. The non-meat protein can comprise a fungal -derived protein, for example, a fungal mycoprotein. The non-meat protein can comprise an insect protein. The meat substitute can comprise an in vitro cultured animal cell.

[0007] The disclosure further provides a plant, animal, or edible mushroom cell comprising an exogenous polynucleotide encoding an RCP polypeptide comprising a sequence at least 80% identical to at least one of SEQ ID NOs:l-12 and comprising an XYG chromophore tripeptide, wherein the RCP polypeptide has an absorption spectrum maximum between 450 nm and 600 nm. The disclosure also provides a meat substitute comprising said cell. The RCP polypeptide can comprise a sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to at least one of SEQ ID NOs: 1-12 and comprises an XYG chromophore tripeptide. The cell can be a plant cell. The cell can be an edible mushroom cell, such as a Fusarium venenatum cell. The cell can be an animal cell. The cell can be an insect cell. The cell can be an in vitro cultured animal cell. The disclosure also provides a meat substitute comprising said cell(s).

[0008] The disclosure also provides a method for increasing the red color of a meat substitute comprising adding a pigment composition comprising an RCP to a non-meat protein to form a meat substitute with increased red color prior to cooking relative to an equivalent meat substitute without the pigment composition. The pigment compositing may be any pigment described herein comprising an RCP. The pigment composition can comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the RCP on a dry weight basis the meat substitute comprises a pigment composition comprising the RCP and the pigment composition comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the RCP on a dry weight basis.

BRIEF DESCRIPTION OF THE FIGURES

[0009] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee.

[0010] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document.

[0011] FIG. l is a photo of E. coli expressing DsRED (top streak) or EforRed (bottom streak) red chromogenic proteins.

[0012] FIG. 2 shows the visible wavelength absorbance data of DsRED (right) and EforRed (left) before and after heating at 80 °C for 20 minutes.

[0013] FIG. 3 shows the visible wavelength absorbance data for wild-type EforRed and the 2-F2 mutant EforRed before and after heating at 80 °C for 20 minutes.

[0014] FIG. 4 shows a series of photos of a small lab scale meat substitute application incorporating a beet juice concentrate, a 2-F2 EforRed mutant, and an EforRed pigment before (top, “raw”) and after (bottom, “cooked”) heating on a hot plate set at 130 ⁰ for 90 seconds.

[0015] FIG. 5 shows a series of photos of a tiny patty meat substitute incorporating a beet juice concentrate, a 2-F2 EforRed mutant, and an EforRed pigment before (top, “raw”) and after (bottom, “cooked”) heating at 80 °C for 20 minutes.

DETAILED DESCRIPTION

[0016] Described herein are pigment compositions for meat substitutes that contain a red chromogenic protein. It has been discovered that red chromogenic proteins may be used in a pigment composition having a similar red/pink color to mimicking animal-based meat products. Meat substitutes containing an effective amount of this pigment composition will transition from a red color when raw to a more brown or less red color when cooked. In an aspect, the brown color results from Maillard reactions involving other components of the meat substitute which may become more visible upon cooking the meat substitute.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. As used herein, each of the following terms has the meaning associated with it as defined below. [0018] As used herein, the terms “meat substitute” and “meat substitute composition” are used interchangeably and refer to compositions that mimic the general appearance, nutritional content, and/or taste of natural animal meat or natural animal meat compositions without containing as the majority component tissues or cells from a whole, living vertebrate animal. For example, the meat substitute may be free of, or contain as a minor component, naturally-occurring animal muscle, adipose, or satellite cells from muscle tissues harvested from a whole vertebrate animal (e.g., a cow, a sheep, a pig, a chicken, a turkey, etc.). In some aspects, the meat substitute is free of any animal cells, e.g., any in vivo derived or in vitro cultured animal cells.

[0019] The meat substitutes and meat substitute compositions described herein include non-meat proteins, plant-based proteins (e.g., pea protein, soy protein, wheat protein, chickpea protein, corn protein, and the like), fungal-based proteins (e.g., mycoproteins derived from fungi such as Fusarium venenatum and the like), in vitro cultured animal cells (e.g., cultured muscle cells, satellite cells, adipose cells, and the like), insect proteins, or combinations thereof. The meat substitute can comprise plant-based proteins including, but not limited to, pea protein, soy protein, wheat protein, chickpea protein, and com protein. The meat substitute can comprise fungal based proteins including, but not limited to, mycoproteins from Fusarium venenatum. The meat substitute can comprise in vitro cultured animal cells including, but not limited to, muscle cells, satellite cells, and adipose cells grown, differentiated and propagated using, for example, fermentation, a bioreactor, scaffold-seeded cell culture, or other artificial methods. The meat substitute can comprise a combination of two or more of plant-based protein, fungal-based proteins, insect proteins and in vitro cultured animal cells. For example, a meat substitute may include a pea protein and a fungal mycoprotein, a soy protein and a cultured bovine muscle cell, a cultured avian adipocyte and a fungal mycoprotein, or any other combination of plant-base protein, fungal-based protein, insect proteins, and in vitro cultured animal cells.

[0020] In some aspects, the meat substitute comprises plant-based proteins, fungal-based proteins, or combinations thereof and is free of any animal-based proteins or cells. In some aspects, the meat substitute comprises plant-based proteins, fungal-based proteins, insect proteins, and combinations thereof and is free of and any vertebrate animal-based cells or proteins. In some aspects, the meat substitute comprises plant-based proteins and is free of fungal-based, insect, or animal-based cells or proteins. In some aspects, the meat substitutes comprises fungal-based proteins and is free of plant-based, insect, and animal-based cells and proteins. In aspect, the meat substitute comprises insect proteins and is free of plant-based, fungal-based, and animal-based cells and proteins. In some aspects, the meat substitute comprises in vivo cultured animal cells and is free of plant-based proteins, fungal-based proteins, insect proteins, and in vivo whole animal derived tissues, cells, and proteins.

[0021] In some aspects, the meat substitute can mimic a beef product, e.g., ground beef, steak, beef jerky, beef ribs, beef patties, beef sausages, and the like. In some aspects, the meat substitute can mimic a pork product, e.g., ground pork, pork chops, ham, smoked pork, bacon, pork sausage, pork patties, pork ribs, and the like. In some aspects, the meat substitute can mimic a chicken product, e.g., ground chicken, chicken breast, check legs, chicken thighs, chicken wings, chicken patties, chicken tenders, chicken nuggets, chicken sausage, and the like. In some aspects, the meat substitute can mimic a turkey product, e.g., ground turkey, turkey sausage, turkey patties, and the like. In some aspects, the meat substitute can mimic a shellfish product, e.g., crab, lobster, shrimp, crayfish, clams, scallops, oysters, mussels, and the like. In some aspects, the meat substitute can mimic a cured, salted, or processed meat product, e.g., charcuterie, salami, summer sausage, prosciutto, bologna, kielbasa, and the like.

[0022] As used herein, the term “non-meat protein” refers to protein sourced from plants, fungus, insects, dairy products, or in vitro cultured animal cells, and excludes in vivo vertebrate animal derived tissues, cells, or proteins. For example, non-meat proteins may include plant-based proteins, fungal-based proteins, insect proteins, milk proteins (e.g., casein and whey), proteins from in vitro cultured animal cells, or combinations thereof.

[0023] As used herein, the terms “red chromogenic protein” (“RCP”) and “pink chromogenic protein” (“PCP”) are used interchangeably and refer to polypeptides which, when correctly folded and, if necessary, in the presence of required co-factors, have an absorbance spectrum maximum between 450 nm and 600 nm. The absorbance spectrum maximum is also referred to in the art as a lambda max. When in an aqueous solution at a concentration of at least 0.5 mg/ml, an RCP appears red or pink when viewed by the naked eye. RCP may also be referred to in the art as “red fluorescent proteins.” RCPs suitable for use in the pigments and meat substitutes described herein include, but are not limited to, cnidarian RCPs, for example, from a species selected from the group consisting of Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. SSAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, and Zoanthus sp. SAL-2001. [0024] The RCP polypeptides described herein are characterized by the chromophore forming tripeptide X-Y-G. Upon spontaneous cyclization and oxidation of the X-Y-G tripeptide, the chromophore includes a phenol ring derived from the Tyr residue of the tripeptide and an N- acylimine formed by desaturation of the Ca-N double bond derived from the Try backbone and formation of a double bond between Ca and Ob of the Tyr. Chromophore formation and how the RCP chromophore differs from, for example, green and blue chromophores are known and described in the art. See, for example, Miyawaki et al. (“Red fluorescent proteins: chromophore formation and cellular applications,” Current Opinion in Structural Biology, 2012, 22:679-688). [0025] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties. As used herein, “enzyme” or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function. A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.

[0026] Table 1: Amino Acid three and one letter symbols

[0027] As used herein, the term “thermostable RCP” refers to an RCP polypeptide that, when heated at 80 °C for 20 minutes, retains at least 80% absorbance at its absorbance spectrum maximum as compared to the absorbance at the absorbance spectrum maximum prior to heating. For example, EforRed has an absorbance spectrum maximum at 580 nm and upon heating at 80 °C for 20 minutes, the heated EforRed retains at least 80% of the absorbance at 580 nm compared to the absorbance prior to heating. In some aspects, the absorbance after heating can be greater than 100% of the absorbance prior to heat. In other words, the thermostable RCP may have a higher absorbance at its absorbance spectrum maximum after heating than it did before.

[0028] As used herein, “EforRed” refers to the RCP from the coral Echinopora forskaliana. GenBank ID EU498726.1. The wild-type polypeptide sequence of EforRed is provided in SEQ ID NO:l and the MYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 1 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the MYG tripeptide of residues 62-64 of SEQ ID NO: 1.

[0029] SEQ ID NO: 1 MSVIKQVMKTKLHLEGTVNGHDFTIEGKGEGKPYEGLQHMKMTVTKGAPLPFSVHILT P SHMY GSKPFNKYP ADIPD YHKO SFPEGMS WERSMIFEDGGVCT ASNHS SFNLOENCFI Y DVKFHGVNLPPDGPVMQKTIAGWEPSVETLYVRDGMLKSDTAMVFKLKGGGHHRVDF KTTYKAKKPVKLPEFHFVEHRLELTKHDKDFTTWDQQEAAEGHFSPLPKALP [0030] As used herein, “meffRed” refers to the RCP from the coral Montipora efflorescens. GenBank ID DQ206379.1. The wild-type polypeptide sequence for meffRed is provided in SEQ ID NO:2 and the DYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:2 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the DYG tripeptide of residues 67-69 of SEQ ID NO:2.

[0031] SEQ ID NO:2

MALSKNGLTKNMTTKYRMEGCVDGHKFVITGDGIGDPFEGKQTSIDLCVVEGGPLPF SE DILSAVFDYGNRVFTKYPODLVDYFKNSCPAGYTWORSFLFEDGAVCTASADITVSVEE NCFYHESKFHGVNFPADGPVMKKMTTNWEPSCEKITPIPNEGILKGDVTMFLLLKDGGR YRCQFDTVYKAKSDPKTIMMPDWHFIQHKLNREDRSDAKHQKWRLVENAIAYRSTLS [0032] As used herein, “asPink” refers to the RCP from Ammonia sulcate. GenBank ID EF587182.1. The wild-type polypeptide sequence for asPink is provided in SEQ ID NO:3 and the MYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 3 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the MYG tripeptide of residues 63-65 of SEQ ID NO:3.

[0033] SEQ ID NO:3:

M ASFLKKTMPFKTTIEGT VN GH YFKC T GKGEGNPFEGT QEMKIE VIEGGPLPF AFHIL S T SCMYGSKTFD YVSGIPDYFKOSFPEGFTWERTTTYEDGGFLTAHODTSLDGDCLVYKV KILGNNFP ADGP VMQNK AGRWEP ATEI VYEVDGVLRGQ SLMALKCPGGRHLTCHLHTT YRSKKP AS ALKMPGFHFEDHRIEIMEEVEKGKC YKQ YE AAV GRY CD AAP SKLGHN [0034] As used herein, “spi sPink” refers to the RCP from Stylophora pistillata. GenBank ID DQ206398. The wild-type polypeptide sequence for spisPink is provided in SEQ ID NO:4 and the KYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:4 and including an XYG chromophore tripeptide, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the KYG tripeptide of residues 66-68 of SEQ ID NO:4.

[0035] SEQ ID NO:4:

MSHSKQ AL ADTMKMTWLMEGS VN GHAFTIEGEGT GKP YEGKQ SGTFRVTKGGPLPF A FDIVAPTLKYGFKCFMKYPADIPDYFKLAFPEGLTYDRKIAFEDGGCATATVEMSLKGN TLVHKTNFQGGNFPIDGPVMQKRTLGWEPTSEKMTPCDGIIKGDTIMYLMVEGGKTLK CRYENNYRANKP VLMPP SHF VDLRLTRTNLDKEGL AFKLEE Y AVARVLE V [0036] As used herein, “DsRed” refers to the RCP from Discosoma sp. GenBank ID AF168419. The wild-type polypeptide sequence for DsRed is provided in SEQ ID NO:5 and the QYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:5 and including an XYG chromophore, said RCP haivng an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the QYG tripeptide of residues 66-68 of SEQ ID NO:5. [0037] SEQ ID NO: 5:

MRS SKN VIKEFMRFK VRMEGT VN GHEFEIEGEGEGRP YEGHNT VKLK VTKGGPLPF AW DILSPOFOYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTODSSLODG CFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHKALKLKDGGHYL VEFK SI YM AKKP V QLPGY Y Y VD SKLDIT SHNED YTIVEQ YERTEGRHHLFL [0038] As used herein, “amilRFP” refers to the RCP from Acropora millepora. GenBank ID AY646073. The wild-type polypeptide sequence for amilRFP is provided in SEQ ID NO:6 and the DYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 6 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the DYG tripeptide of residues 66-68 of SEQ ID NO:6.

[0039] SEQ ID NO:6:

MALSKHGLTKDMTMKYHMEGSVDGHKFVITGHGNGNPFEGKQTMNLCVVEGGPLPFS EDILSAAFDYGNRVFTEYPOGMVDFFKNSCPAGYTWHRSLLFEDGAVCTTSADITVSVE ENCFYHNSKFHGVNFPADGPVMKKMTTNWEPSCEKIIPVPRQGILKGDIAMYLLLKDGG RYRCQFDTIYKAKSDPKEMPEWHFIQHKLTREDRSDAKNQKWQLVEHAVASRSALPG [0040] As used herein, “ceriantRFP” refers to the RCP from Cerianthus sp. GenBank ID AY296063. The wild-type polypeptide sequence for ceriantRFP is provided in SEQ ID NO:7 and the QYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 7 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the QYG tripeptide of residues 61-63 of SEQ ID NO:7.

[0041] SEQ ID NO:7:

MNLSKNV S V S VYMKGNVNNHEFEYDGEGGGDP YTGKY SMKMTLRGQNCLPF S YDIIT T AFO Y GFRVF TK YPEGI VD YFKD SLPD AF O WNRRI VFEDGGVLNM S SPIT YKDNVLHGD VW A V GVNFPPN GP VMKNEI VMEEPTEETF TPKN GVL V GF CPK A YLLKDGS Y Y Y GNMT TF YRSKKSGQ APPGYHF VKHRL VKTN V GHGFKTVEQTEY AT AHV SDLPK [0042] As used herein, “dis2RFP” refers to the RCP from Discosoma sp. SSAL-2000. GenBank ID AF272711. The wild-type polypeptide sequence for dis2RFP is provided in SEQ ID NO:8 and the QYG chromophore tripeptide is underline. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 8 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore tripeptide is the QYG tripeptide of residues 66-68 of SEQ ID NO:8.

[0043] SEQ ID NO: 8:

MSCSKNVIKEFMRFKVRMEGTVNGHEFEIKGEGEGRPYEGHCSVKLMVTKGGPLPFA F DILSPOF O Y GSKVYVKHP ADIPDYKKLSFPEGFKWERVMNFEDGGVVT VSODS SLKDG CFIYEVKFIGVNFP SDGP VMQRRTRGWE AS SERLYPRDGVLKGDIHMALRLEGGGHYL VEFKSIYMVKKPSVQLPGYYYVDSKLDMTSHNEDYTVVEQYEKTQGRHHPFIKPLQ [0044] As used herein, “eqFP611” refers to the RCP from Entacmaea quadricolor. GenBank ID AY130757. The wild-type polypeptide sequence of eqFP611 is provided in SEQ ID NO:9 and the MYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 9 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore is the MYG chromophore of residues 63-65 of SEQ ID NO:9.

[0045] SEQ ID NO:9:

MNSLIKENMRMMVVMEGSVNGYQFKCTGEGDGNPYMGTQTMRIKVVEGGPLPFAFDI L AT SFM Y GSKTFIKHTKGIPDFFKO SFPEGF T WER VTRYEDGGVF T VMOD T SLEDGCL V YFLAK VT GVNFP SN GA VMQKKTKGWEPNTEML YP ADGGLRGY S QM ALNVDGGGYL S C SFETTYRSKKTVENFKMPGFHFVDHRLERLEESDKEMFVVQHEHAVAKFCDLPSKLGR L

[0046] As used herein, “KO” refers to the RCP from Lithophyllon concinna. GenBank ID AB128820. The wild-type polypeptide sequence of KO is provided in SEQ ID NO: 10 and the CYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 10 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore is the CYG chromophore of residues 64-66 of SEQ ID NO: 10.

[0047] SEQ ID NO: 10:

MSVIKPEMKMKYFMDGSVNGHEFTVEGEGTGKPYEGHQEMTLRVTMAKGGPMPFSFD L V SHTF C Y GHRPFTKYPEEIPDYFKO AFPEGLSWERSLOFEDGGF AAV S AHISLRGNCFE HKSKFVGVNFPADGPVMQNQSSDWEPSTEKITTCDGVLKGDVTMFLKLAGGGNHKCQ FKTTYKAAKKILKMPQSHFIGHRLVRKTEGNITELVEDAVAHC

[0048] As used herein, “pporRFP” refers to the RCP from Porites porites. GenBank ID DQ206380. The wild-type polypeptide sequence for pporRFP is provided in SEQ ID NO: 11 and the MYG chromophore is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 11 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore is the MYG chromophore of residues 67-69 of SEQ ID NO: 11.

[0049] SEQ ID NO:l l:

MAL SKQ SGVKD VMNTELHMDGIVNGHPFEIKGKGKGNP YKGVQTMKLT VIKGAPLPF S IDILLPOHMY GSKPFIKYPESIPD YIKLSFPEGITWERSMTFEDGAV CTV SND SRLDGD SFI YE VRFLGVNFPRDGP VMQKKTRGWDP S TERL YEC GGW QRGD VHM ALKLEN GGH YT C DFKTT YK SKKGLK VPP YHF VDHKLDLL SHNTDGATFEEFEQREI AHAHL SNLP V A [0050] As used herein, “zoan2RFP” refers to the RCP from Zoanthus sp. SAL-2001. GenBank ID AY059642. The wild-type polypeptide sequence of zoan2RFP is provided in SEQ ID NO: 12 and the DYG chromophore tripeptide is underlined. A pigment or meat substitute composition as described herein may include an RCP polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 12 and including an XYG chromophore, said RCP having an absorption spectrum maximum between 450 nm and 600 nm. As indicated below, the XYG chromophore is the DYG chromophore of residues 66-68 of SEQ ID NO: 12.

[0051] SEQ ID NO: 12:

MAHSKHGLTDDMTMHFRMEGCVDGHKFVIEGNGNGNPFKGKQFINLCVIEGGPLPFS E DILSAAFDYGNRLFTEYPEGIVDYFKNSCPAGYTWHRSFRFEDGAVCICSADITVNVREN CIYHESTF Y GVNFP ADGPVMKKMTTNWEPSCEKIIPIN SQKILKGD V SMYLLLKDGGRY RCQFDTIYKAKTEPKEMPDWHFIQHKLNREDRSDAKNQKWQLIEHAIASRSALP [0052] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed pigments, compositions, and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability to exhibit the desired activity. Generally, the variant or modified sequence may include or greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the wild-type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein. [0053] As used herein, the phrases “% sequence identity,” “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well-known. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0054] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0055] The polypeptides disclosed herein may include “variant” polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild-type” is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant, “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0056] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. “Conservative amino acid substitutions” are those substitutions that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge and/or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. [0057] As used herein, terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides or any fragment thereof. There phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. The DNA polynucleotides may be a cDNA or a genomic DNA sequence.

[0058] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0059] Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotides (i.e., polynucleotides encoding an EforRed polypeptide) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in coral are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.

[0060] Also provided herein are polynucleotides encoding an RCP described herein. The polynucleotide may encode any of the RCP polypeptides described herein, for example, the polynucleotide may encode a polypeptide with a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to any one of SEQ ID NOs: 1-12 and an absorbance spectrum maximum between 450 nm and 600 nm.

[0061] The polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding the thermolabile EforRed polypeptide. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.

[0062] Cells including any of the polynucleotides, constructs, or vectors described herein are also provided. The cell may be a procaryotic cell or a eukaryotic cell. Suitable procaryotic cells include bacteria cell, for example, Escherichia coli and Bacillus subtilis cells. Suitable eukaryotic cells include, but are not limited to, fungal cells, plant cells, and animal cells. Suitable fungal cells include, but are not limited to, Fusarium venenatum , Pichia pastoris , Saccharomyces cerevisiae, Kluyveromyces lactis , Yarrow ia lipolytica , Trichomderma reesei, Issatchenkia orientalis , and Aspergillus niger cells. Suitable plant cells include, but are not limited to, a pea cell ( Pisum sativum ), a corn cell (Zea mays), a soybean cell ( Glycine max), and a wheat cell ( Triticum sp.). Suitable animal cells include, but are not limited to, muscle cells (e.g., myocytes, myoblasts, myosatellite, and satellite cells) and fat cells (e.g., adipocytes or adipocyte progenitor cells such as mesenchymal stem cells). Suitable animal cells may be mammalian (e.g., bovine, porcine, and ovine), avian (e.g., poultry), crustacean (e.g., shrimp, lobster, and crab), mollusk (e.g., clam, mussel, scallop, and oyster) or insect cells. In some aspects, the cell is an edible mushroom cell, which refers to a mushroom that is safe for human consumption. For example, the edible mushroom cell can be a Fusarium venenatum , Agaricus bisporus, Lentinula edodes, or Volvariella volvacea cell.

[0063] Described herein are pigment compositions containing an RCP polypeptide, and meat substitutes including such pigment compositions. The pigment compositions disclosed herein can be used to provide color to a meat substitute that is similar to the color of natural animal meat when raw. The pigment composition may provide a pink and/or red color to a raw, uncooked meat substitute. The pigment composition may provide a pink and/or red color to a cured meat product. [0064] The pigment compositions described herein include an RCP. Suitable RCPs are described herein and include, but are not limited to cnidarian RCPs, for example, an RCP from Montipora efflorescens , Echinopora forskalicma , Ammonia sulcate , Stylophora pistillata, Discosoma sp., Acropora millepora, Cerianthus sp., Discosoma sp. SSAL-2000, Entacmaea quadricolor, Lithophyllon concinna, Porites porites, or Zoanthus sp. SAL-2001. The pigment composition may include a polypeptide at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs:l-12 and an absorption spectrum maximum between 450 nm and 600 nm.

[0065] The pigment composition can be included in a meat substitute at a level that provides increased or improved pink and/or red color in the meat substitute, while also providing increased or improved brown color in the meat substitute after cooking. In an aspect, the pigment composition is used at a level of at least 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 1.25%, or 1.5% on a wet (total) weight basis in a meat substitute composition. The pigment composition may be used at a level in the range of 0.01% to 6%, 0.05% to 5%, 0.1% to 3%, or 0.5% to 2% by weight in a meat substitute composition.

[0066] The pigment composition may additionally include a carrier or a diluent. The pigment composition may also include a blend of the RCP polypeptide with another color or pigment. For example, the pigment composition may include the RCP polypeptide and a fruit or vegetable extract-based pigment composition. [0067] The pigment composition described herein can be used as a pigment in any meat substitute composition. An exemplary, but non-limiting, meat substitute composition is a composition which comprises: plant protein (e.g., textured pea protein and/or pea protein), water, vegetable oil, flavor ingredients, salt, sugar, binders, and the pigment composition described herein. The pigment composition described herein can also be used in food applications other than meat substitutes. [0068] Meat substitutes described herein may include one or more cells comprising an exogenous polynucleotide encoding an RCP as described herein. For example, the meat substitutes may include a fungal, plant, or animal cell as described herein comprising an exogenous polynucleotide encoding an RCP described herein.

[0069] Also provided is a method for increasing the red or pink color of a meat substitute. The method for increasing the red color of a meat substitute may include adding a pigment composition comprising an RCP to a non-meat protein to form a meat substitute with increased red color prior to cooking relative to an equivalent meat substitute without the pigment composition. Suitable RCPs include, but are not limited to, any RCP described herein. For example, the RCP may be a polypeptide at least 80% identical to at least one of SEQ ID NOs: 1-12.

[0070] Also provided is a method for increasing the red color of a cured meat substitute. The method for increasing the color of a cured meat substitute may include adding a pigment composition comprising an RCP to a non-meat protein to form a cured meat substitute with increased red color prior relative to an equivalent cured meat substitute without the pigment composition. Suitable RCPs include, but are not limited to, any RCP described herein. For example, the RCP may be a polypeptide at least 80% identical to at least one of SEQ ID NOs: 1-12.

EXAMPLES

[0071] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Example 1 : EforRed and DsRed thermostability [0072] EforRed was expressed in E. coli cells using a His6 tag with a protease cleavage site (MGS SHHHHHHS SGLVPRGSH, SEQ ID NO: 13) on the N-terminus of the EforRed sequence of SEQ ID NO:l. Both red chromogenic proteins EforRed and DsRed are red/pink in color (FIG. 1) and demonstrate the corresponding absorbance peak at the 580 nm wavelength. Upon heating at 80 °C for 20 minutes, both EforRed and DsRed remained stable, maintaining the absorbance peak and 580 nm, with DsRed showing an increase in absorbance at 580 nm following heating (FIG. 2).

Example 2: Application tests of RCP pigment compositions

[0073] EforRed was expressed in E. coli as outlined in Example 1. The 2-F2 mutant was mutagenized in E. coli using the expression system outlined in Example 1. In a small-scale application model of a meat substitute composition, each of the beet juice concentrate, the EforRed 2-F2 mutant, and the wild-type EforRed pigments increased the red color of the raw meat substitute composition (FIG. 4). Upon heating on a hot plate at 130 °C for 90 seconds to simulate cooking on a skillet (see “Cooked” in FIG. 4), the red color of the EforRed 2-F2 mutant pigment was reduced while the beet juice and wild-type EforRed pigments retained the red color. Hunter colorimetry data for each of meat substitute pigmentations are reported in Table 3.

[0074] The color change in the small-scale application model occurred from the surface towards the interior, mimicking “rare internal appearance observed when the cooking animal-derived ground beef. Other pigments showed no fading (e.g. beet juice concentrate) or consistent fading (e.g. deglycosylated beet juice, not shown) throughout the entire product (FIG. 5).

Table 3: Hunter colorimetry of pigment containing meat substitutes