Methods and compositions for enhanced expression of exogenous caseins in transgenic plants
AssigneeMozza Foods, Inc.
InventorCory Tobin
Life of this patent
Estimating the term…
Abstract
The present disclosure provides methods and plants by genetically modifying plants to enhance their nutritional quality. In some aspects, the present disclosure provides methods for reducing activity or expression of one or more proteases in a plant (e.g., soybean), leading to increased levels of casein proteins and casein micelles, resulting in plants with enhanced levels of essential amino acids and improved digestibility.
Claims141
A mutated alpha casein protein represented by any of the sequences identified in SEQ I.D. Nos. 199-204.
A mutated alpha casein protein, wherein the mutated alpha casein protein comprises at least 80% of any of the sequences identified in SEQ ID Nos. 199-204.
A mutated beta-casein protein represented by any of the sequences identified in SEQ ID Nos. 207-212.
A mutated beta-casein protein, wherein the mutated beta-casein protein comprises at least 80% of any of the sequences identified in SEQ. ID Nos. 207-212.
A mutated kappa casein represented by DNA sequence identified in SEQ ID No. 215 and Amino Acid Sequence identified in SEQ ID No. 216.
A method for promoting the expression of a casein protein in a plant, comprising inhibiting an expression or activity of a protease in the plant, wherein a reduced activity or expression of the protease increases the expression of the casein protein in the plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising A) expressing the casein protein in the transgenic plant;
- and B) decreasing proteasomal activity or expression in the transgenic plant to decrease degradation of the casein protein expressed in the transgenic plant.
A soy plant, wherein the soy plant:
- 1) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and 2) has reduced proteasomal activity or expression level compared to a wildtype soy plant.
A transgenic soy plant comprising a casein gene, wherein the transgenic soy plant exhibits reduced expression of the ubiquitin/26S proteasome compared to a non-transgenic soy plant, and wherein the reduced expression comprises reduced or no expression levels of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), E3 (ubiquitin ligase), or any combination thereof.
A method for increasing casein protein expression in a transgenic soy plant, the method comprising:
- A) expressing the casein protein in the transgenic plant;
- and B) reducing the activity or expression of the ubiquitin/26S proteasome pathway in the soy plant or part thereof to increase the expression of the casein protein in the transgenic plant;
- wherein the reducing the expression comprises reduced or no expression levels of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), E3 (ubiquitin ligase), or any combination thereof.
A transgenic soy plant, wherein the transgenic soy plant expresses a casein protein, and wherein the soy plant has increased activity or expression level of a deubiquitinating enzyme compared to a wildtype soy plant.
A method for expressing a casein protein in a transgenic soy plant, the method comprising A) expressing the casein protein in the transgenic plant;
- and B) increasing the expression level of a deubiquitinating enzyme in the soy plant, such that the soy plant has increased expression of the casein protein compared to without increasing the expression level of the deubiquitinating enzyme.
A method for increasing a casein protein expression in a transgenic soy plant, the method comprising A) expressing the casein protein in the transgenic plant;
- and B) enhancing the stability and prolonging the half-life of the casein protein in the soy plant through the use of chemical post-translational modifications (PTMs) comprising nitrosylation, methylation, acetylation, lipidation, or pegylation.
A method for increasing casein protein expression in a transgenic soy plant, the method comprising A) expressing the casein protein in the transgenic plant;
- and B) conjugating the casein protein to a cross-linker, wherein the cross-linker is selected from the group consisting of Genipin, Ethylenediamine, Cystamine, Bis(sulfosuccinimidyl) suberate (BS3), or 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Glutaraldehyde, ethylene glycol diglycidyl ether (EGDE), polyethylene glycol diglycidyl ether (PEGDE), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate (DSS), 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP), bis[sulfosuccinimidyl] suberate (BS3), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), formaldehyde, dimethyl adipimidate (DMA), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), genipin, ethylenediamine, cystamine, bismaleimidohexane (BMH), disuccinimidyl tartrate (DST), ethylene glycol bis[succinimidylsuccinate] (EGS), N,N′-ethylenebis(iodoacetamide) (EBI), N,N′-(1,3-phenylene)dibutyric acid di[succinimidyl ester] (DSS), N,N′-(ethylene-di-1,2-phenylene)bismaleimide, N,N′-1,4-phenylenebismaleimide, divinyl sulfone, diisopropylcarbodiimide, dicyclohexylcarbodiimide, diethylcarbodiimide, difluorodinitrobenzene, N,N′-disuccinimidyl carbonate (DSC), dithiobispropionimidate, glyoxal, 1,5-difluoro-2,4-dinitrobenzene, N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIA), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and succinimidyl 6-[(beta-maleimidopropionamido)hexanoate] (SMPH).
A casein molecule comprising a casein protein and a cross-linker, wherein the wherein the cross-linker is selected from the group consisting of Genipin, Ethylenediamine, Cystamine, Bis(sulfosuccinimidyl) suberate (BS3), or 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Glutaraldehyde, ethylene glycol diglycidyl ether (EGDE), polyethylene glycol diglycidyl ether (PEGDE), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate (DSS), 3,3′-dithiobis[sulfosuccinimidylpropionate] (DTSSP), bis[sulfosuccinimidyl] suberate (BS3), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), formaldehyde, dimethyl adipimidate (DMA), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), genipin, ethylenediamine, cystamine, bismaleimidohexane (BMH), disuccinimidyl tartrate (DST), ethylene glycol bis[succinimidylsuccinate] (EGS), N,N′-ethylenebis(iodoacetamide) (EBI), N,N′-(1,3-phenylene)dibutyric acid di[succinimidyl ester] (DSS), N,N′-(ethylene-di-1,2-phenylene)bismaleimide, N,N′-1,4-phenylenebismaleimide, divinyl sulfone, diisopropylcarbodiimide, dicyclohexylcarbodiimide, diethylcarbodiimide, difluorodinitrobenzene, N,N′-disuccinimidyl carbonate (DSC), dithiobispropionimidate, glyoxal, 1,5-difluoro-2,4-dinitrobenzene, N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIA), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and succinimidyl 6-[(beta-maleimidopropionamido)hexanoate] (SMPH).
A method for increasing casein protein expression in a transgenic soy plant, the method comprising mutating the casein protein to comprise fewer Proline (P), Glutamic acid (E), Serine(S), or Threonine (T) compared to a wildtype casein protein.
A fusion protein, comprising a casein protein and a peptide sequence that represents a ubiquitin-associated (UBA) domain.
A method for increasing casein protein expression in a transgenic soy plant;
- the method comprising expressing a casein protein in the transgenic soy plant, wherein the casein protein is fused with a peptide sequence that represents a ubiquitin-associated (UBA) domain.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising expressing a casein protein in the transgenic soy plant, wherein the transgenic plant comprises a promoter that regulates the expression of the casein gene in the transgenic plant, and wherein the promoter is non-naturally occurring.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) has a modified promoter of the casein gene compared to a wildtype soy plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising optimizing the codons of the casein gene in the plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) has optimized codons of the casein gene compared to a wildtype soy plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant cell, comprising expressing the casein protein in the transgenic plant cell;
- wherein the transgenic plant comprises at least two, at least three, at least four, at least five, at least six copies, at least seven copies, or at least eight copies of DNA sequences coding the casein gene in the transgenic plant cell.
A soy plant cell, wherein the soy plant cell expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein, wherein the soy plant cell comprises at least two, at least three, at least four, at least five, at least six copies, at least seven copies, or at least eight copies of DNA sequences coding the casein gene in the soy plant cell.
A method for enhancing translation of a casein protein in a transgenic plant, comprising one or more of expressing the casein protein in the transgenic plant, wherein the transgenic plant comprises a mRNA coding the casein protein;
- and wherein the mRNA is post-transcriptionally modified to increase the translation of the casein protein.
A soy plant, wherein the soy plant expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and wherein the soy plant comprises a mRNA coding the casein protein;
- and wherein the mRNA is post-transcriptionally modified to increase the translation of the casein protein.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising mutating the sequence of the casein protein in the plant to increase the expression levels and stability of a casein protein in a transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) has a mutated sequence of the casein protein compared to a wildtype casein protein.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising editing the chromosomal location of the casein gene in the plant to increase the expression levels of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) has a different chromosomal location of the casein gene.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising using a chaperone protein to assist in the folding and assembly of the casein protein in the plant, such that the transgenic plant has increased expression levels and stability of the casein protein.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises a chaperone protein to assist in the folding and assembly of the casein protein.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising incorporating a translational enhancer in the sequence of the casein gene in the plant to increase the expression levels and stability of a casein protein in a transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises a translational enhancer incorporated in the sequence of the casein gene.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising adding a Scaffold/Matrix Attachment Region (SAR or Mar) to the casein gene in the plant to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises a Scaffold/Matrix Attachment Region (SAR or Mar) added to the casein gene.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising incorporating introns into the coding sequence of the casein gene in the plant to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises introns incorporated into the coding sequence of the casein gene to increase the expression levels and stability of the casein protein in the transgenic plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising inducing hormonal changes in the plant to enhance casein gene expression.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) wherein the soy plant has undergone hormonal induction to enhance casein gene expression.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising using antisense RNAs to suppress negative regulators of casein expression in the plant, to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises an antisense RNA to suppress negative regulators of casein expression.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising modifying the 3′ Untranslated Region (UTR) of the casein gene in the plant, to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises a modified 3′ Untranslated Region (UTR) of the casein gene.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising optimizing the signal peptide of the casein protein in the plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) comprises an optimized signal peptide of the casein protein compared to a wildtype soy plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising fusing the casein protein with another protein in the plant, to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant expresses a fusion of the casein protein with another protein compared to a wildtype soy plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising knocking down protease activity or expression in the plant to increase the expression levels and stability of the casein protein in the transgenic plant.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) has reduced protease activity or expression level compared to a wildtype soy plant.
A method for enhancing expression levels and stability of a casein protein in a transgenic plant, comprising optimizing the nutrient and environmental conditions of the plant to enhance casein gene expression.
A soy plant, wherein the soy plant:
- A) expresses a casein protein comprising alpha-S1-casein, alpha-S2-casein, beta-casein or kappa-casein;
- and B) is grown under optimized nutrient and environmental conditions to enhance casein gene expression compared to a wildtype soy plant.
A method of expressing casein proteins in a plant, wherein the method results in increased expression of the casein gene in the soy plant or part thereof compared to a soy plant or part thereof not subjected to the method.
A casein protein modified by lipidation, wherein the lipidation is cysteine prenylation, N-terminal glycine myristoylation, cysteine palmitoylation, or serine and lysine fatty acylation.
A modified casein protein, comprising a casein protein and a polyethylene glycol (PEG), wherein the polyethylene glycol (PEG) is covalently attached to casein protein.
A method of extracting casein proteins from the soybean plant;
- comprising providing using a buffer comprising a protease inhibitor selected from the group consisting of a Serine Protease Inhibitor, a Cysteine Protease Inhibitor, an Aspartic Protease Inhibitor, a Metalloprotease Inhibitor, a Covalent Protease Inhibitor, a Reversible Protease Inhibitor, a Peptide-based Protease Inhibitor, a Synthetic Protease Inhibitor, or a Natural Protease Inhibitor.
A soybean seed comprising casein proteins, wherein the casein proteins comprise at least 0.03% of the total weight of the seed, at least 0.041% of the total weight of the seed, or at least 0.055% of the total weight of the seed, or at least 0.075% of the total weight of the seed, or at least 0.101% of the total weight of the seed, or at least 0.136% of the total weight of the seed, or at least 0.184% of the total weight of the seed, or at least 0.248% of the total weight of the seed, or at least 0.335% of the total weight of the seed, or at least 0.452% of the total weight of the seed, or at least 0.610% of the total weight of the seed, or at least 0.824% of the total weight of the seed, or at least 1.113% of the total weight of the seed, or at least 1.503% of the total weight of the seed, or at least 2.031% of the total weight of the seed, or at least 2.744% of the total weight of the seed, or at least 3.707% of the total weight of the seed, or at least 5.006% of the total weight of the seed, or at least 6.761% of the total weight of the seed, or at least 13% of the total weight of the seed.
A soybean seed comprising casein proteins, wherein the casein proteins comprise at least 0.075% of the total protein content of the seed, or at least 0.103% of the total protein content of the seed, or at least 0.138% of the total protein content of the seed, or at least 0.188% of the total protein content of the seed, or at least 0.253% of the total protein content of the seed, or at least 0.340% of the total protein content of the seed, or at least 0.460% of the total protein content of the seed, or at least 0.620% of the total protein content of the seed, or at least 0.838% of the total protein content of the seed, or at least 1.130% of the total protein content of the seed, or at least 1.525% of the total protein content of the seed, or at least 2.060% of the total protein content of the seed, or at least 2.783% of the total protein content of the seed, or at least 3.758% of the total protein content of the seed, or at least 5.078% of the total protein content of the seed, or at least 6.860% of the total protein content of the seed, or at least 9.267% of the total protein content of the seed, or at least 12.503% of the total protein content of the seed, or at least 16.903% of the total protein content of the seed, or at least 32.5% of the total protein content of the seed.
A soybean seed comprising casein micelles, wherein the casein micelles comprise at least 0.03% of the total weight of the seed, or at least 0.041% of the total weight of the seed, or at least 0.055% of the total weight of the seed, or at least 0.075% of the total weight of the seed, or at least 0.101% of the total weight of the seed, or at least 0.136% of the total weight of the seed, or at least 0.184% of the total weight of the seed, or at least 0.248% of the total weight of the seed, or at least 0.335% of the total weight of the seed, or at least 0.452% of the total weight of the seed, or at least 0.610% of the total weight of the seed, or at least 0.824% of the total weight of the seed, or at least 1.113% of the total weight of the seed, or at least 1.503% of the total weight of the seed, or at least 2.031% of the total weight of the seed, or at least 2.744% of the total weight of the seed, or at least 3.707% of the total weight of the seed, or at least 5.006% of the total weight of the seed, or at least 6.761% of the total weight of the seed, or at least 13% of the total weight of the seed.
A soybean seed comprising casein micelles, wherein the casein micelles comprise at least 0.075% of the total protein content of the seed, or at least 0.103% of the total protein content of the seed, or at least 0.138% of the total protein content of the seed, or at least 0.188% of the total protein content of the seed, or at least 0.253% of the total protein content of the seed, or at least 0.340% of the total protein content of the seed, or at least 0.460% of the total protein content of the seed, or at least 0.620% of the total protein content of the seed, or at least 0.838% of the total protein content of the seed, or at least 1.130% of the total protein content of the seed, or at least 1.525% of the total protein content of the seed, or at least 2.060% of the total protein content of the seed, or at least 2.783% of the total protein content of the seed, or at least 3.758% of the total protein content of the seed, or at least 5.078% of the total protein content of the seed, or at least 6.860% of the total protein content of the seed, or at least 9.267% of the total protein content of the seed, or at least 12.503% of the total protein content of the seed, or at least 16.903% of the total protein content of the seed, or at least 32.5% of the total protein content of the seed.
A method for enhancing translational efficiency in genetically modified organism, comprising the use of both 5′ and 3′ untranslated regions (UTRs) within a single genetic construct.
A genetic construct designed for enhanced translational efficiency, comprising both a 5′ UTR and a 3′ UTR.
A method for enhancing the stability of transgenic casein in soy plants, comprising expressing a broad-spectrum protease inhibitor within said soy plants.
A soy plant genetically modified to express transgenic casein and a broad-spectrum protease inhibitor, wherein the protease inhibitor provides protection against proteolytic degradation.
A plasmid construct for use in transforming soy plants to express transgenic casein, the construct comprising a broad-spectrum protease inhibitor gene and a casein gene, each under the control of a constitutive promoter.
A method for producing soy-based products with enhanced casein content, comprising cultivating the soy plant of claim 106 and harvesting the soy plant material containing the stabilized transgenic casein.
A method for inhibiting protease activity in transgenic soy plants expressing casein, comprising the step of downregulating endogenous proteases using RNA interference technology.
A transgenic soy plant with increased casein stability, wherein the plant has reduced activity or expression of proteases through the introduction of a broad-spectrum protease inhibitor gene.
A kit for transforming soy plants to produce stable transgenic casein, comprising a vector with a broad-spectrum protease inhibitor gene, a casein gene, and instructions for use in a plant transformation protocol.
A method for producing transgenic casein in soy plants for use in cheese production, the method comprising expressing a bovine casein gene alongside a broad-spectrum protease inhibitor in the soy plant.
A soy plant with reduced expression of endogenous proteases, produced by a method comprising the steps of transforming the soy plant with a plasmid containing a broad-spectrum protease inhibitor gene and cultivating the transformed plant under conditions that promote the expression of the inhibitor and casein proteins.
A method for modifying endogenous protease in a plant to enhance the expression of transgenic casein.
A plant comprising a modified protease and a transgenic casein.
A method for producing a modified thiol proteinase with reduced ability to cleave intracellular proteins.
A method for enhancing the expression of foreign casein genes in soybeans, comprising the steps of co-expressing said casein genes with Bowman Birk Protease Inhibitors (BBPIs) in a soybean plant, wherein said BBPIs are serine protease inhibitors that reduce the degradation of said casein proteins, thereby increasing their expression within the plant.
A transgenic plant comprising 1) a reduced level of protease activity or expression, as compared to a wild-type counterpart;
- and 2) an increased levels of casein, as compared to a counterpart without the reduced level of protease activity or expression.
Dependencies are inferred from the claim text.