Application US20210040512

RECOMBINANT PRODUCTION SYSTEMS FOR PRENYLATED POLYKETIDES OF THE CANNABINOID FAMILY
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US20210040512

1. A modified recombinant host cell comprising: (i) a first exogenous polynucleotide that encodes an acyl-CoA synthetase that converts an aliphatic carboxylic acid to an acyl CoA thioester, wherein the acyl-CoA synthetase is a revS polypeptide or a CsAAE3 polypeptide; (ii) a second exogenous polynucleotide that encodes olivetolic acid synthase, (iii) a third exogenous polynucleotide that encodes a 2-alkyl-4,6-dihydroxybenzoic acid cyclase; (iv) a fourth exogenous polynucleotide that encodes a prenyltransferase that catalyzes coupling of geranyl-pyrophosphate to a 2-alkyl-4,6-dihydroxybenzoic acid to produce an acidic cannabinoid; and (8) (4)

one or more additional exogenous polynucleotides selected from:

an exogenous polynucleotide that encodes a prenol and isoprenol kinase;

an exogenous polynucleotide that encodes a kinase that produces dimethylallyl pyrophosphate and isopentenyl pyrophosphate when grown in the presence of exogenous prenol and isoprenol; and

an exogenous polynucleotide that encodes a geranyl-pyrophosphate synthase.

2. The modified recombinant host cell of claim 1, wherein the 2-alkyl-4,6-dihydroxybenzoic acid cyclase is a truncated olivetolic acid cyclase. (0)

3. The modified recombinant host cell of claim 1, wherein one or more of the exogenous polynucleotides is present in an autonomously replicating expression vector. (1) (0)

5. The modified recombinant host cell of claim 1, the exogenous polynucleotides are present in two or more autonomously replicating expression vectors. (0)

6. The modified recombinant host cell of claim 1, wherein the modified recombinant host cell is a yeast cell genetically modified to knockout expression of the PAD1 and FDC1 aromatic decarboxylase genes. (0)

7. The modified recombinant host cell of claim 1, wherein one or more of the exogenous polynucleotides are integrated into the host genome. (0)

8. The modified recombinant host cell of claim 1, wherein expression of one or more of the exogenous polynucleotides is driven by an ADH2 promoter, an ADH1 promoter, a GAL1 promoter, a MET25 promoter, a CUP1 promoter, a GPD promoter, a PGK promoter, a PYK promoter, a TPI promoter, or a TEF1 promoter. (0)

9. The modified recombinant host cell of claim 1, wherein the host cell is a cell selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha and Aspergillus. (0)

20. A method of producing a cannabinoid product, the method comprising culturing the modified host cell of claim 1 under conditions in which products encoded by the exogenous polynucleotides are expressed and the acidic cannabinoid is produced. (0)

10. A modified recombinant host cell comprising: (i) a first exogenous polynucleotide that encodes prenol and isoprenol kinase; (ii) a second exogenous polynucleotide that encodes kinase activity to produce dimethylallyl pyrophosphate and isopentenyl pyrophosphate when grown in the presence of exogenous prenol and isoprenol; (iii) a third exogenous polynucleotide that encodes a geranyl-pyrophosphate synthase; and (iv) a fourth exogenous polynucleotide that encodes a prenyltransferase that catalyzes coupling of geranyl-pyrophosphate to a 2-alkyl-4,6-dihydroxybenzoic acid to form an acidic cannabinoid. (5) (0)

11. The modified recombinant host cell of claim 10, wherein one or more of the first, second, third, or fourth exogenous polynucleotides is present in an autonomously replicating expression vector. (3) (0)

15. The modified recombinant host cell of claim 10, wherein the 2-alkyl-4,6-dihydroxybenzoic acid is expressed within the host cell and the host cell further comprises a fifth exogenous polynucleotide encoding an olivetolic acid synthase gene, a sixth exogenous polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoic acid cyclase; and a seventh exogenous polynucleotide encoding an acyl-CoA synthase gene. (1) (0)

17. The modified recombinant host cell of claim 10, further comprising an exogenous polynucleotide that encodes a cannabinoid synthase enzyme that catalyzes conversion of the acidic cannabinoid compound intermediate produced in the host cell to form a neutral cannabinoid or a second acidic cannabinoid. (0)

18. The modified recombinant host cell of claim 10, wherein the host cell is a cell selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha and Aspergillus. (0)

19. The modified recombinant host cell of claim 10, wherein one or more of the exogenous polynucleotides is operably linked to an ADH2 promoter, an ADH1 promoter, a GAL1 promoter, a MET25 promoter, a CUP1 promoter, a GPD promoter, a PGK promoter, a PYK promoter, a TPI promoter, or a TEF1 promoter. (0)

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Application US20210040512

RECOMBINANT PRODUCTION SYSTEMS FOR PRENYLATED POLYKETIDES OF THE CANNABINOID FAMILY

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