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Application 20200291434
Inmed Pharmaceuticals Inc.

Metabolic Engineering Of E. Coli For The Biosynthesis Of Cannabinoid Products

Provided herein are methods and compositions for producing terpenoids in a host cell. In some cases, the terpenoids are cannabinoids.

Much More than Average Length Specification


1 Independent Claims

  • Claim 1. An expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDF enzyme.
  • Claim 11. A plasmid comprising an expression set according to any one of claims 1-10.
  • Claim 21. A host cell comprising any one of the expression cassettes of claims 1-10 and/or any one of the plasmids of claims 11-20.
  • Claim 22. A host cell comprising: a. an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDF enzymeand b. an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a terpenoid synthase.
  • Claim 32. The host cell of any one of claims 22-31, wherein the cell is a prokaryote.
  • Claim 33. The host cell of any one of claims 22-32, wherein the expression cassette of a) and/or b) is integrated into the genome of the host cell.
  • Claim 34. The host cell of any one of claims 22-33, wherein the host cell comprises a nucleic acid encoding a cannabinoid synthase operably linked to a promoter and the promoter operably linked to the nucleic acid encoding the cannabinoid synthase is a constitutive promoter.
  • Claim 35. The host cell of claims 22-33, wherein the host cell comprises a nucleic acid encoding a cannabinoid synthase operably linked to a promoter and the promoter operably linked to the nucleic acid encoding the cannabinoid synthase is an inducible promoter.
  • Claim 36. The host cell of any one of claims 22-35, wherein the expression cassette of a) and the expression cassette of b) are present in a single plasmid or inserted into a genome of the host cell at a single locus.
  • Claim 37. The host cell of any one of claims 22-35, wherein the expression cassette of a) and the expression cassette of b) are in different plasmids or inserted into a genome of the host cell at different loci.
  • Claim 38. The host cell of any one of claims 22-37, wherein the host cell further comprises olivetolic acid (OA).
  • Claim 40. The host cell of any one of claims 22-39, wherein the host cell comprises an expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding one or more glycosylation pathway genes
  • Claim 41. The host cell of any one of claims 22-40, wherein the host cell comprises a deletion in 1, 2, 3, 4, 5, 6, or all of the genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA.
  • Claim 42. A method of obtaining a target metabolic product, the method comprising culturing a host cell according to any one of claims 22-41 in a suitable culture medium under conditions suitable to induce expression in one or more host cell expression cassettes, and then harvesting the cultured cells or spent medium, thereby obtaining the target metabolic product.
  • Claim 46. The method of any one of claims 42-45, wherein the method comprises harvesting and lysing the cultured cells, thereby producing cell lysate.
  • Claim 48. The method of any one of claims 42-45, wherein the method comprises purifying the target metabolic product from the spent culture medium, thereby producing a purified target metabolic product.