IP Library › Granted Patent US 12,385,072
Granted Patent B2
US 12,385,072 · App. 17/962,229 · Granted Aug 12, 2025

Bidirectional multi-enzymatic scaffolds for biosynthesizing cannabinoids

Inventor: Jordan Buck (Boulder, CO)
Assignee: Khona Scientific Holdings, Inc.
C12P7/42C12N15/70C12N15/74C12N15/81C12N15/8222C12N2330/51C12N2800/40C12Y101/01034C12Y101/01157C12Y103/01038C12Y103/03C12Y121/03007C12Y121/03008C12Y203/01009C12Y203/01016C12Y203/01206C12Y203/03008C12Y203/0301C12Y205/01001C12Y207/01036C12Y207/04002C12Y401/01033C12Y402/01017C12Y404/01026C12Y503/03002C12Y604/01002
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Quick Facts
Patent No.
US 12,385,072
App. No.
17/962,229
Granted
Aug 12, 2025
Kind
B2
Abstract

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

Claims (41)

1. A host cell capable of producing one or more cannabinoids, 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 acetyl-CoA acetyltransferase activity and comprising a seventh heterologous interaction domain,

(h) an eighth exogenous nucleic acid encoding an eighth polypeptide having ATP citrate lyase activity and comprising an eighth heterologous interaction domain,

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

(j) a tenth exogenous nucleic acid encoding a tenth polypeptide having isopentyl-diphosphate isomerase activity and comprising a tenth heterologous interaction domain,

(k) an eleventh exogenous nucleic acid encoding an eleventh polypeptide having diphospho-mevalonate decarboxylase activity and comprising an eleventh heterologous interaction domain,

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

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

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

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

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

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

wherein each peptide ligand for each of said first to fifteenth 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, and (7) said peptide ligand for said eighth 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 ninth heterologous interaction domain, (2) said peptide ligand for said tenth heterologous interaction domain, (3) said peptide ligand for said eleventh heterologous interaction domain, (4) said peptide ligand for said twelfth heterologous interaction domain, (5) said peptide ligand for said thirteenth heterologous interaction domain, (6) said peptide ligand for said fourteenth heterologous interaction domain, (7) said peptide ligand for said fifteenth heterologous interaction domain, (8) said peptide ligand for said seventh heterologous interaction domain, and (9) said peptide ligand for said eighth heterologous interaction domain.

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

3. 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.

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

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

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

7. The host cell of claim 6 , wherein said bacterial cell is selected from the group consisting of Escherichia coli, Bacillus, Brevibacterium, Streptomyces , and Pseudomonas cells.

8. The host cell of claim 6 , wherein said yeast cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus , and Komagataella phaffii cells.

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

10. The host cell of claim 9 , wherein said algae is Dunaliella sp., Chlorella variabilis, Euglena mutabilis , or Chlamydomonas reinhardtii cells.

11. The host cell of claim 9 , wherein said plant cell is a Cannabis or tobacco cell.

12. The host cell of claim 1 , wherein each of said polypeptides is of the formula: enzyme-linker1-spacer-linker2-motifi-linker3-motif2, wherein linker1, linker2, and linker3 are the same or different, wherein motif1 and motif2 are the same or different, and wherein motif1 and motif2 form said heterologous interaction domain.

13. The host cell of claim 12 , wherein said scaffold polypeptide comprises a linker between each adjacent peptide ligand.

14. The host cell of claim 13 , wherein said scaffold polypeptide is tagged with a MYC tag, FLAG tag, or HA tag.

15. The host cell of claim 12 , wherein said linker is a flexible GS-rich sequence flanking a rigid α-helical moiety.

16. The host cell of claim 12 , wherein said spacer is the cTPR6 spacer.

17. 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 sixteenth exogenous nucleic acid encoding said polypeptide scaffold.

18. 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 sixteenth exogenous nucleic acid encoding said polypeptide scaffold.

19. The host cell of claim 18 , wherein said constitutive promoter used to express said polypeptide scaffold has weaker constitutive activity level than said constitutive promoter used to express said polypeptides.

20. 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.

21. The host cell of claim 20 , wherein said promoter is the GAL1-10 promoter.

Assignments (5)
SECURITY INTEREST Recorded May 20, 2025
From: KHONA SCIENTIFIC HOLDINGS, INC.
To: ORLADY, PAUL; SHORTAL, JOSEPH
Reel/Frame 071173/0398 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 2, 2024
From: BUCK, JORDAN
To: KHONA PHARMS LLC
Reel/Frame 066977/0044 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 2, 2024
From: KHONA PHARMS LLC
To: KHONA SCIENTIFIC LLC
Reel/Frame 066977/0048 →
COMPANY CONVERSION FROM LLC TO INCORPORATION Recorded Apr 2, 2024
From: KHONA SCIENTIFIC, LLC
To: KHONA SCIENTIFIC, INC.
Reel/Frame 066977/0554 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 2, 2024
From: KHONA SCIENTIFIC, INC.
To: KHONA SCIENTIFIC HOLDINGS, INC.
Reel/Frame 066977/0569 →
Continuity (4)
Continuation 16694417 · Nov 25, 2019
Provisional Application 62836265 · Apr 19, 2019
Provisional Application 62771839 · Nov 27, 2018
Related Publication 20230265465A1 · Aug 24, 2023
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