Generation Of Water-Soluble Cannabinoids Utilizing Protein Cannabinoid-Carriers
The inventive technology includes novel systems, methods, and compositions for the generation of water-soluble short-chain fatty acid phenolic compounds, preferably cannabinoids, terpenes, and other volatile compounds produced in Cannabis . In particular, the inventive technology includes novel systems, methods, and compositions to solubilize short-chain fatty acid phenolic coin-pounds, such as cannabinoids, via binding to a water soluble and readily digested carrier protein such as: lipocalins, lipocalin-like, odorant-binding proteins, and odorant-binding-like proteins.
1 - 14 . (canceled)
15 . A solubilized cannabinoid composition comprising:
a carrier protein having a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure bound to at least one cannabinoid to form a water-soluble protein-cannabinoid complex.
16 . The composition of claim 15 , wherein the carrier protein comprises a carrier protein having an amino acid sequence selected from the group of consisting of: SEQ ID NOs. 1-46, and 113-148, or a homolog having affinity towards at least one cannabinoid thereof.
17 . (canceled)
18 . The composition of claim 15 , wherein the carrier protein is coupled with a secretion signal.
19 . The composition of claim 18 , wherein said secretion signal comprises a secretion signal having an amino acid sequence selected from the group consisting of: SEQ ID NO. 47, and SEQ ID NOs. 106-112.
20 . The composition of claim 15 , wherein the at least one cannabinoid comprises a cannabinoid selected from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBDA), Δ 9 -tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabigerol (CBG), and cannabigerolic acid CBGA).
21 . The composition of claim 15 , wherein said carrier protein having affinity towards at least one cannabinoid comprises an Olfactory Binding Protein (OBP)-carrier protein having a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure, or Lipocalin Cannabinoid (LC)-carrier protein having a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure.
22 - 46 . (canceled)
47 . A method of solubilizing a cannabinoid comprising the steps of:
generating a Lipocalin Carrier (LP)-carrier protein having affinity towards at least one cannabinoid; and
introducing said LC-carrier protein to said at least one cannabinoid, wherein said LC-carrier protein binds said at least one cannabinoid to form a water-soluble protein-cannabinoid complex.
48 . The method of claim 47 , wherein the LC-carrier protein comprises an LC-carrier protein having an amino acid sequence selected from the group of consisting of: SEQ ID NOs. 1-29, and 30-46, or a homolog having affinity towards at least one cannabinoid thereof.
49 . (canceled)
50 . The method of claim 47 , wherein the LC-carrier protein is coupled with a secretion signal.
51 - 52 . (canceled)
53 . The method of claim 47 , wherein the at least one cannabinoid comprises a cannabinoid selected from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBDA), Δ 9 -tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabigerol (CBG), and cannabigerolic acid (CBGA).
54 . The method of claim 47 , wherein said LC-carrier protein having affinity towards at least one cannabinoid comprises an LC-carrier protein having a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure.
55 . The method of claim 47 , wherein the LC-carrier comprises an engineered LC-carrier protein having a truncated LC-carrier protein forming a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure.
56 . The method of claim 55 , wherein said truncated LC-carrier protein comprises an truncated LC-carrier protein having an amino acid sequence selected from the group of consisting of: SEQ ID NOs. 30-46.
57 - 61 . (canceled)
62 . A method of solubilizing a cannabinoid comprising the steps of:
establishing a cell culture of genetically modified yeast, plant, or bacteria cells that express a nucleotide sequence, operably linked to a promoter, encoding a heterologous Lipocalin Carrier (LC)-carrier protein wherein said heterologous LC-carrier protein exhibits affinity towards one or more cannabinoids;
introducing one or more cannabinoids to the genetically modified yeast, plant, or bacteria cell culture; and
binding said LC-carrier protein with said one or more cannabinoids to form a water-soluble protein-cannabinoid complex;
wherein said LC-carrier protein includes a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure.
63 - 64 . (canceled)
65 . The method of claim 62 , wherein said heterologous LC-carrier protein comprises a heterologous LC-carrier protein having an amino acid sequence selected from the group of consisting of: SEQ ID NOs. 1-29, and 30-46, or a homolog having affinity towards at least one cannabinoid thereof.
66 - 68 . (canceled)
69 . The method of claim 62 , wherein the at least one cannabinoid comprises a cannabinoid selected from the group consisting of: cannabidiol (CBD), cannabidiolic acid (CBDA), Δ 9 -tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabigerol (CBG), and cannabigerolic acid (CBGA).
70 . The method of claim 62 , and further comprising the of step of genetically modifying the LC-carrier protein to form an engineered LC-carrier protein having enhanced affinity for at least one cannabinoid, such genetic modification comprising at least one of the following:
replacing one or more amino acid residues of the LC-carrier protein cannabinoid binding pocket with side chains orientated toward the binding cavity;
replacing one or more amino acid residues of the LC-carrier protein cannabinoid binding pocket having a hydrophilic side chain with amino acid residues having a hydrophobic side chain; and
replacing one or more small hydrophobic amino acid residues of the LC-carrier protein cannabinoid binding pocket with larger hydrophobic amino acid residues.
71 - 72 . (canceled)
73 . (canceled)
74 . The method of claim 62 , wherein the LC-carrier comprises an engineered LC-carrier protein further comprising a truncated LC-carrier protein forming a β-barrel enclosed cannabinoid-binding site having an internal cavity, and an external loop scaffold structure.
75 . The method of claim 74 , wherein said truncated LC-carrier protein comprises an truncated LC-carrier protein having an amino acid sequence selected from the group of consisting of: SEQ ID NOs. 30-46.
76 - 87 . (canceled)
70 . The method of claim 15 , and further comprising the of step of genetically modifying the LC-carrier protein to form an engineered LC-carrier protein having enhanced affinity for at least one cannabinoid, such genetic modification comprising at least one of the following:
replacing one or more amino acid residues of the LC-carrier protein cannabinoid binding pocket with side chains orientated toward the binding cavity;
replacing one or more amino acid residues of the LC-carrier protein cannabinoid binding pocket having a hydrophilic side chain with amino acid residues having a hydrophobic side chain; and
replacing one or more small hydrophobic amino acid residues of the LC-carrier protein cannabinoid binding pocket with larger hydrophobic amino acid residues.