Systems, methods, and compositions for the generation of water-soluble cannabinoids utilizing fatty-acid binding proteins and fatty-acid binding-like proteins as 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 and other cannabinoid producing plants. In particular, the inventive technology includes novel systems and methods to solubilize short-chain fatty acid phenolic compounds, such as cannabinoids, via binding to a water soluble and readily digested carrier protein.
1. A method of solubilizing a cannabinoid comprising the steps of:
modifying a Fatty-Acid Binding Protein (FABP)-carrier protein having affinity towards a cannabinoid to produce a recombinant FABP-carrier protein having enhanced affinity towards said cannabinoid;
introducing said recombinant FABP-carrier protein to said cannabinoid, wherein said recombinant FABP-carrier protein binds said cannabinoid forming a water-soluble protein-cannabinoid complex; and
wherein the recombinant FABP-carrier protein comprises a recombinant FABP-carrier protein having the amino acid sequence selected from the group consisting of: SEQ ID NOs. 79-86, and wherein said cannabinoid is cannabidiol (CBD), or D 9 -tetrahydrocannabinol (THC).
2. The method of claim 1 , wherein said step of genetically modifying the FABP-carrier protein comprises at least one of the following:
replacing one or more amino acid residues of the FABP-carrier protein cannabinoid binding pocket with side chains orientated toward the binding cavity;
replacing one or more amino acid residues of the FABP-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 FABP-carrier protein cannabinoid binding pocket with larger hydrophobic amino acid residues.
3. The method of claim 1 , wherein the recombinant FABP-carrier protein is expressed in a protein production system selected from the group consisting of:
a bacterial cell culture;
a yeast cell culture;
a plant cell culture;
a fungi cell culture;
an algae cell culture;
a bioreactor production system; and
a plant.
4. The method of claim 1 , wherein the recombinant FABP-carrier protein is coupled with a secretion signal.
5. The method of claim 4 , wherein said secretion signal is selected from the group consisting of: SEQ ID NO. 3, SEQ ID NO. 65, and SEQ ID NOs. 69-73.
6. The method of claim 1 , wherein said recombinant FABP-carrier protein is further genetically modified to decrease aggregation propensity and potential antigenicity.
7. The method of claim 6 , wherein said recombinant FABP-carrier protein comprises a recombinant FABP-carrier protein having one or both of the following mutations: V58E, and V42R.
8. A method of solubilizing a cannabinoid comprising the steps of:
expressing in a protein production system a heterologous nucleotide sequence, operably linked to a promoter, encoding generating a Fatty-Acid Binding Protein (FABP)-carrier protein having affinity towards a cannabinoid; and
introducing said FABP-carrier protein to said cannabinoid, wherein said FABP-carrier protein binds said cannabinoid to form a water-soluble protein-cannabinoid complex, wherein the FABP-carrier protein comprises a FABP-carrier protein having the amino acid sequence selected from the group of consisting of: SEQ ID NOs. 79-86, or a sequence having at least 95% or more sequence identity with any of the SEQ ID NOs. 79-86, and wherein said cannabinoid is cannabidiol (CBD), or Δ 9 -tetrahydrocannabinol (THC).
9. The method of claim 8 , wherein the recombinant FABP-carrier protein is expressed in a protein production system selected from the group consisting of:
a bacterial cell culture;
a yeast cell culture;
a plant cell culture;
a fungi cell culture;
an algae cell culture;
a bioreactor production system; and
a plant.
10. The method of claim 8 , wherein said recombinant FABP-carrier protein comprises a recombinant FABP-carrier protein having one or both of the following mutations: V58E, and V42R.