IP Library › Granted Patent US 11,525,148

Granted Patent B2

US 11,525,148 · App. 16/694,417 · Granted Dec 13, 2022

Bidirectional multi-enzymatic scaffolds for biosynthesizing cannabinoids

Inventor: Jordan Buck (Boulder, CO)

Assignee: Khona Scientific Holdings, Inc.

C12P7/42 C12N15/70 C12N15/74 C12N15/81 C12N15/8222 C12N2330/51 C12N2800/40 C12Y101/01034 C12Y101/01157 C12Y103/01038 C12Y103/03 C12Y121/03007 C12Y121/03008 C12Y203/01009 C12Y203/01016 C12Y203/01206 C12Y203/0301 C12Y203/03008 C12Y205/01001 C12Y207/01036 C12Y207/04002 C12Y401/01033 C12Y402/01017 C12Y404/01026 C12Y503/03002 C12Y604/01002

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Quick Facts

Patent No.: US 11,525,148
App. No.: 16/694,417
Granted: Dec 13, 2022
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

Abstract

This document relates to using bidirectional, multi-enzymatic scaffolds to biosynthesize cannabinoids in recombinant hosts.

Claims (68)

1. A host cell capable of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabidiolic acid, and cannabichromenic acid, said host cell comprising:

(a) a first exogenous nucleic acid encoding a first polypeptide having CBGA synthase activity and comprising a first heterologous interaction domain,

(b) a second exogenous nucleic acid encoding a second polypeptide having olivetolic acid cyclase activity and comprising a second heterologous interaction domain,

(c) a third exogenous nucleic acid encoding a third polypeptide having olivetol synthase activity and comprising a third heterologous interaction domain,

(d) a fourth exogenous nucleic acid encoding a fourth polypeptide having trans-2-enoyl-CoA reductase activity and comprising a fourth heterologous interaction domain,

(e) a fifth exogenous nucleic acid encoding a fifth polypeptide having enoyl-CoA hydratase activity and comprising a fifth heterologous interaction domain,

(f) a sixth exogenous nucleic acid encoding a sixth polypeptide having 3-hydroxybutyryl-CoA dehydrogenase activity and comprising a sixth heterologous interaction domain,

(g) a seventh exogenous nucleic acid encoding a seventh polypeptide having beta-ketothiolase activity and comprising a seventh heterologous interaction domain,

(h) an eighth exogenous nucleic acid encoding an eighth polypeptide having acetyl-CoA acetyltransferase activity and comprising an eighth heterologous interaction domain,

(i) a ninth exogenous nucleic acid encoding a ninth polypeptide having ATP citrate lyase activity and comprising a ninth heterologous interaction domain,

(j) a tenth exogenous nucleic acid encoding a tenth polypeptide having geranyl pyrophosphate synthase activity and comprising a tenth heterologous interaction domain,

(k) an eleventh exogenous nucleic acid encoding an eleventh polypeptide having isopentyl-diphosphate isomerase activity and comprising an eleventh heterologous interaction domain,

(l) a twelfth exogenous nucleic acid encoding a twelfth polypeptide having diphospho-mevalonate decarboxylase activity and comprising a twelfth heterologous interaction domain,

(m) a thirteenth exogenous nucleic acid encoding a thirteenth polypeptide having phosphomevalonate kinase activity and comprising a thirteenth heterologous interaction domain,

(n) a fourteenth exogenous nucleic acid encoding a fourteenth polypeptide having mevalonate kinase activity and comprising a fourteenth heterologous interaction domain,

(o) a fifteenth exogenous nucleic acid encoding a fifteenth polypeptide having HMG-CoA reductase activity and comprising a fifteenth heterologous interaction domain,

(p) a sixteenth exogenous nucleic acid encoding a sixteenth polypeptide having HMG-CoA synthase activity and comprising a sixteenth heterologous interaction domain, and

(q) a seventeenth exogenous nucleic acid encoding a polypeptide scaffold comprising a peptide ligand for each of said first to sixteenth heterologous interaction domains,

wherein each of said first to sixteenth heterologous interaction domains is different,

wherein each peptide ligand for each of said first to sixteenth heterologous interaction domains is different,

wherein said polypeptide scaffold comprises, in an order extending in a first direction away from said peptide ligand for said first heterologous interaction domain, (1) said peptide ligand for said second heterologous interaction domain, (2) said peptide ligand for said third heterologous interaction domain, (3) said peptide ligand for said fourth heterologous interaction domain, (4) said peptide ligand for said fifth heterologous interaction domain, (5) said peptide ligand for said sixth heterologous interaction domain, (6) said peptide ligand for said seventh heterologous interaction domain, (7) said peptide ligand for said fourth heterologous interaction domain, (8) said peptide ligand for said fifth heterologous interaction domain, (9) said peptide ligand for said sixth heterologous interaction domain, (10) said peptide ligand for said eighth heterologous interaction domain, and (11) said peptide ligand for said ninth heterologous interaction domain, and

wherein said polypeptide scaffold comprises, in an order extending in the other direction away from said peptide ligand for said first heterologous interaction domain, (1) said peptide ligand for said tenth heterologous interaction domain, (2) said peptide ligand for said eleventh heterologous interaction domain, (3) said peptide ligand for said twelfth heterologous interaction domain, (4) said peptide ligand for said thirteenth heterologous interaction domain, (5) said peptide ligand for said fourteenth heterologous interaction domain, (6) said peptide ligand for said fifteenth heterologous interaction domain, (7) said peptide ligand for said sixteenth heterologous interaction domain, (8) said peptide ligand for said eighth heterologous interaction domain, and (9) said peptide ligand for said ninth heterologous interaction domain.

2. The host cell of claim 1 , wherein each of said polypeptides is of the formula: enzyme-linker 1 -spacer-linker 2 -motif 1 -linker 3 -motif 2 , wherein linker 1 , linker 2 , and linker 3 are the same or different, wherein motif 1 and motif 2 are the same or different, and wherein motif 1 and motif 2 form said heterologous interaction domain.

3. The host cell of claim 2 , wherein said scaffold polypeptide comprises a linker between each adjacent peptide ligand.

4. The host cell of claim 3 , wherein said scaffold polypeptide is tagged with a MYC tag, FLAG tag, or HA tag.

5. The host cell of claim 2 , wherein said linker is a flexible GS-rich sequence flanking a rigid α-helical moiety.

6. The host cell of claim 2 , wherein said spacer is the cTPR6 spacer.

7. The host cell of claim 1 , wherein a constitutive promoter is operably linked to one or more of said exogenous nucleic acids encoding said polypeptides or to said seventeenth exogenous nucleic acid encoding said polypeptide scaffold.

8. The host cell of claim 1 , wherein each said exogenous nucleic acid comprises an inducible promoter operably linked to the sequence encoding said polypeptide or said polypeptide scaffold.

9. The host cell of claim 8 , wherein said promoter is the GAL1-10 promoter.

10. The host cell of claim 1 , wherein a first constitutive promoter is operably linked to one or more of said exogenous nucleic acids encoding said polypeptides and a second constitutive promoter is operably linked to said seventeenth exogenous nucleic acid encoding said polypeptide scaffold.

11. The host cell of claim 10 , wherein said constitutive promoter used to express said polypeptide scaffold has weaker constitutive activity level than said constitutive promoter used to express said polypeptides.

12. The host cell of claim 1 , further comprising (r) an eighteenth exogenous nucleic acid encoding an acetyl-CoA carboxylase and comprising an eighteenth heterologous interaction domain, and (s) a nineteenth exogenous nucleic acid encoding a polypeptide scaffold comprising a peptide ligand for each of said ninth and eighteenth heterologous interaction domains.

13. The host cell of claim 1 , wherein said host cell further comprises an exogenous nucleic acid encoding a cannabidiolic acid synthase and a cannabichromenic acid synthase.

14. The host cell of claim 1 , wherein said host cell further comprises an exogenous cannabidiolic acid synthase.

15. The host cell of claim 1 , wherein said host cell further comprises an exogenous cannabichromenic acid synthase.

16. The host cell of claim 1 , wherein said host cell is a bacterial or a yeast host cell.

17. The host cell of claim 16 , wherein said bacterial cell is selected from the group consisting of Escherichia coli, Bacillus, Brevibacterium, Streptomyces , and Pseudomonas cells.

18. The host cell of claim 16 , wherein said yeast cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus , and Komagataella phaffii cells.

19. The host cell of claim 1 , wherein said host cell is an algae or a plant cell.

20. The host cell of claim 19 , wherein said algae is Dunaliella sp., Chlorella variabilis, Euglena mutabilis , or Chlamydomonas reinhardtii cells.

21. The host cell of claim 19 , wherein said plant cell is a Cannabis or tobacco cell.

22. A method of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabidiolic acid, and cannabichromenic acid, said method comprising culturing a host cell under conditions wherein said host cell produces said one or more cannabinoids, wherein said host cell comprises: