Biosynthetic platform for the production of cannabinoids and other prenylated compounds

1. 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.

2. The recombinant, artificial, or engineered metabolic pathway of claim 1 , wherein the recombinant, artificial, or engineered metabolic pathway is in a cell-free system.

3. The recombinant, artificial, or engineered metabolic pathway of claim 1 , wherein the first cofactor-dependent enzyme comprises dehydrogenase activity using NAD+ as the cofactor.

4. The recombinant, artificial, or engineered metabolic pathway of claim 1 , wherein the enzyme that recycles the cofactor is an NAD (P) H oxidase.

5. The recombinant, artificial, or engineered metabolic pathway of claim 1 , wherein the recombinant, artificial, or engineered metabolic pathway comprises one or more of the following enzymes:

(i) hexokinase (Hex);

(ii) Glucose-6-phosphate isomerase (Pgi);

(iii) Phosphofructokinase (Pfk);

(iv) Fructose-1,6-bisphosphate aldolase (Fba);

(v) Triose phosphate isomerase (Tpi);

(vi) Gald-3-P dehydrogenase (Gap);

(vii) a mutant Gald-3-P dehydrogenase (mGap);

(viii) NADH Oxidase (Nox)

(ix) Phosphoglycerate Kinase (Pgk)

(x) Phosphoglycerate Mutase (2,3 BPG dependent or Mn2+dependent) (dPgm or iPgm);

(xi) Enolase (eno);

(xii) Pyruvate Kinase (FBP dependent/pykF or AMP dependent/pykA);

(xiii) Pyruvate Oxidase (PyOx);

(xiv) Acetyl-phosphate transferase (PTA);

(xv) Acetyl-CoA acetyltransferase (PhaA);

(xvi) HMG-COA Synthase (HMGS);

(xvii) HMG-COA Reductase (HMGR);

(xviii) Mevalonate Kinase (MVK);

(xix) Phosphomevalonate Kinase (PMVK);

(xx) Diphosphomevalonate decarboxylase (MDC); and

(xxi) Geranyl-PP synthase (GPPS) or Farnesyl-PP synthease mutant S82F.

6. The recombinant, artificial, or engineered metabolic pathway of claim 1 , wherein the polypeptide having prenyltransferase activity converts 1-geranyl pyrophosphate to a prenyl-compound when a suitable substrate is present.

7. The recombinant, artificial, or engineered metabolic pathway of claim 6 , wherein the suitable substrate is selected from the group consisting of apigenin, olivetolic acid, divarinic acid, resveratrol, and 2,4-dihydroxybenzoic acid or a derivative thereof.

8. The recombinant, artificial, or engineered metabolic pathway of claim 6 , wherein the prenyl-compound is a cannabinoid.

9. 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.

10. The recombinant, artificial, or engineered metabolic pathway of claim 9 , wherein the pathway is contacted with a suitable substrate is selected from the group consisting of apigenin, olivetolic acid, divarinic acid, resveratrol, and 2,4-dihydroxybenzoic acid or a derivative thereof.

11. The recombinant, artificial, or engineered metabolic pathway of claim 10 , wherein the derivative of 2,4-dihydroxy benzoic acid is a compound of Formula I:

wherein R is H, CH 3 , or X, wherein X is selected from a halo, hydroxyl, cyano, nitro, ester, alkoxy, amino, thiol, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanato, isothiocyanato, thial, borono, boronate, phosphate, aldehyde, carboxyl, carboxamido, azido, cyanato, isocyanato, an optionally substituted (C 1 -C 10 )alkyl, an optionally substituted (C 2 -C 10 )alkenyl, an optionally substituted (C 2 -C 10 )alkynyl, an optionally substituted (C 1 -C 10 ) hetero-alkyl, an optionally substituted (C 2 -C 10 ) hetero-alkenyl, an optionally substituted (C 2 -C 10 ) hetero-alkynyl, an optionally substituted (C 3 -C 10 ) cycloalkyl, an optionally substituted aryl, and an optionally substituted heterocycle.

12. The recombinant, artificial, or engineered metabolic pathway of claim 9 , wherein the polypeptide having prenyltransferase activity converts 1-geranyl pyrophosphate to a prenyl-compound selected from a cannabinoid, a flavonoid, or a stilbenoid in the presence of a suitable substrate.

13. The recombinant, artificial, or engineered metabolic pathway of claim 12 , wherein the suitable substrate is olivetolate and the prenyl-compound is cannabigerolic acid (CBGA), wherein the suitable substrate is divarinic acid and the prenyl-compound is cannabigerovarinic acid (CBGVA), or the suitable substrate is 2,4-dihydroxybenzoic acid or a derivative thereof and the prenyl-compound is CBGXA, wherein X is selected from a halo, hydroxyl, cyano, nitro, ester, alkoxy, amino, thiol, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanato, isothiocyanato, thial, borono, boronate, phosphate, aldehyde, carboxyl, carboxamido, azido, cyanato, isocyanato, an optionally substituted (C 1 -C 10 )alkyl, an optionally substituted (C 2 -C 10 )alkenyl, an optionally substituted (C 2 -C 10 )alkynyl, an optionally substituted (C 1 -C 10 ) hetero-alkyl, an optionally substituted (C 2 -C 10 ) hetero-alkenyl, an optionally substituted (C 2 -C 10 ) hetero-alkynyl, an optionally substituted (C 3 -C 10 ) cycloalkyl, an optionally substituted aryl, and an optionally substituted heterocycle.

14. 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.

15. The recombinant, artificial, or engineered metabolic pathway of claim 14 , wherein the suitable substrate is olivetolate and the prenyl-compound is cannabigerolic acid (CBGA), wherein the suitable substrate is divarinic acid and the prenyl-compound is cannabigerovarinic acid (CBGVA), or the suitable substrate is 2,4-dihydroxybenzoic acid or a derivative thereof and the prenyl-compound is CBGXA, wherein X is selected from a halo, hydroxyl, cyano, nitro, ester, alkoxy, amino, thiol, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanato, isothiocyanato, thial, borono, boronate, phosphate, aldehyde, carboxyl, carboxamido, azido, cyanato, isocyanato, an optionally substituted (C 1 -C 10 )alkyl, an optionally substituted (C 2 -C 10 )alkenyl, an optionally substituted (C 2 -C 10 )alkynyl, an optionally substituted (C 1 -C 10 ) hetero-alkyl, an optionally substituted (C 2 -C 10 ) hetero-alkenyl, an optionally substituted (C 2 -C 10 ) hetero-alkynyl, an optionally substituted (C 3 -C 10 ) cycloalkyl, an optionally substituted aryl, and an optionally substituted heterocycle.