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US 12,180,516 B2Granted
Biosynthetic platform for the production of cannabinoids and other prenylated compounds
AssigneeThe Regents of the University of California
InventorsJames U. Bowie, Meaghan Valliere, Tyler P. Korman, Nicholas Woodall
Life of this patent
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Abstract
Provided is an enzyme useful for prenylation and recombinant pathways for the production of cannabinoids, cannabinoid precursors and other prenylated chemicals in a cell free system as well and recombinant microorganisms that catalyze the reactions.
Claims15
A recombinant, artificial, or engineered metabolic pathway, comprising:
- (i) conversion of pyruvate to acetyl phosphate;
- (ii) conversion of acetyl phosphate to acetyl-coA;
- (iii) a first cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-bisphosphoglycerate;
- (iv) a second cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-biphosphoglycerate, wherein the second cofactor-dependent enzyme is mutated to have its cofactor preference altered, wherein the second cofactor-dependent enzyme has dehydrogenase activity and uses NADP+ as a cofactor, and wherein the second cofactor-dependent enzymes has the sequence of SEQ ID NO:37, but has (1) a P191D mutation or (2) D34A/L35R/T36K mutations;
- (v) an enzyme that recycles a cofactor, wherein the cofactor is selected from the group consisting of NAD+/NADH, NADP+/NADPH, and FAD+/FADH;
- and
- (vi) a polypeptide that has prenyltransferase activity, wherein the polypeptide has a sequence that is at least 95% identical to SEQ ID NO:30 and has mutations selected from the group consisting of:
- Y228N;
- Y288A and F213H;
- Y288N and V49S;
- Y288S and V49N;
- Y288A and V49S;
- Y288N and G286S;
- Y288A, F213N and A232S;
- Y288N, F213G, V49T and V271H;
- Y288A and G286S;
- Y288A, G286S and A232S;
- Y288A, G286S, A232S and F213H;
- Y288V and G286S;
- Y288A and A232S;
- and Y288V and A232S.
18 more elements
A recombinant, artificial, or engineered metabolic pathway, comprising:
- (i) conversion of pyruvate to acetyl phosphate;
- (ii) conversion of acetyl phosphate to acetyl-coA;
- (iii) a first cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-bisphosphoglycerate;
- (iv) a second cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-biphosphoglycerate, wherein the second cofactor-dependent enzyme is mutated to have its cofactor preference altered;
- (v) an enzyme that recycles a cofactor, wherein the cofactor is selected from the group consisting of NAD+/NADH, NADP+/NADPH, and FAD+/FADH;
- and
- (vi) a polypeptide that has prenyltransferase activity, wherein the polypeptide has a sequence that is at least 95% identical to SEQ ID NO:30 and has mutations selected from the group consisting of:
- Y228N;
- Y288A and F213H;
- Y288N and V49S;
- Y288S and V49N;
- Y288A and V49S;
- Y288N and G286S;
- Y288A, F213N and A232S;
- Y288N, F213G, V49T and V271H;
- Y288A and G286S;
- Y288A, G286S and A232S;
- Y288A, G286S, A232S and F213H;
- Y288V and G286S;
- Y288A and A232S;
- and Y288V and A232S;
- wherein the recombinant, artificial, or engineered metabolic pathway is in a cell-free system.
19 more elements
A recombinant, artificial, or engineered metabolic pathway, comprising:
- (i) an enzyme that converts pyruvate to acetyl phosphate;
- (ii) an enzyme that converts acetyl phosphate to acetyl-coA;
- (iii) a first cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-bisphosphoglycerate;
- (iv) a second cofactor-dependent enzyme that converts glyceraldehyde-3-phosphate to 1,3,-biphosphoglycerate, wherein the second cofactor-dependent enzyme is mutated to have its cofactor preference altered;
- (v) an enzyme that recycles a cofactor, wherein the cofactor is selected from the group consisting of NAD+/NADH, NADP+/NADPH, and FAD+/FADH;
- and
- (vi) a polypeptide that has prenyltransferase activity, wherein the polypeptide has a sequence that is at least 95% identical to SEQ ID NO:30 and has mutations selected from the group consisting of:
- Y228N;
- Y288A and F213H;
- Y288N and V49S;
- Y288S and V49N;
- Y288A and V49S;
- Y288N and G286S;
- Y288A, F213N and A232S;
- Y288N, F213G, V49T and V271H;
- Y288A and G286S;
- Y288A, G286S and A232S;
- Y288A, G286S, A232S and F213H;
- Y288V and G286S;
- Y288A and A232S;
- and Y288V and A232S and convert 1-geranyl pyrophosphate to a prenyl-compound when a suitable substrate is present;
- wherein the suitable substrate is selected from the group consisting of olivetolic acid, divarinic acid, and 2,4-dihydroxybenzoic acid or a derivative thereof;
- wherein the recombinant, artificial, or engineered metabolic pathway is in a cell-free system.
20 more elements
Dependencies are inferred from the claim text.