Biosynthetic platform for the production of cannabinoids and other prenylated compounds

1. A recombinant polypeptide comprising a sequence selected from the group consisting of:

(a) SEQ ID NO:30 and having at least a Y288X mutation, wherein X is A, N, S or V;

(b) SEQ ID NO:30 having at least a Y288X mutation, wherein X is A, N, S or V, and at least one other mutation selected from V49Z 1 , F213Z 2 , A232S, I234T, V271Z 3 and/or G286S, wherein Z 1 is S, N, T or G, Z 2 is H, N or G and Z 3 is N or H;

(c) a sequence of (a) or (b) further comprising from 1-20 conservative amino acid substitutions and having prenyltransferase (NphB) activity; and

(d) a sequence that is at least 95%, 98% or 99% identical to SEQ ID NO:30 and which has at least the mutations of (a) or (b);

wherein the polypeptide of (a)-(d) can perform prenylation reactions.

2. The recombinant polypeptide of claim 1 , wherein the polypeptide comprises SEQ ID NO:30 and has a mutation selected from the group consisting of:

(i) V288A;

(ii) Y288N;

(iii) Y288N and F213H;

(iv) Y288A and F213N;

(v) Y288N and V49S,

(vi) Y288S and V49N;

(vii) Y288A and V49S,

(viii) Y288N and G286S;

(ix) Y288N, F213N and V49G;

(x) Y288A, F213N and I234T,

(xi) Y288S, F213N and V49N;

(xii) Y288A, F213N and A232S;

(xiii) Y288N, F213G and V49T,

(xiv) Y288N, F213N, V49S and V271N;

(xv) Y288N, F213G, V49T and V2711-1;

(xvi) Y288A and G286S;

(xvii) Y288A, G286S and A232S;

(xviii) Y288A, G286S, A232S and F213H;

(xix) Y288V and G286S;

(xx) Y288A and A232S; and

(xxi) Y288V and A232S.

3. The recombinant polypeptide of claim 1 having a sequence of SEQ ID NO:30 and having Y288A and G286S mutations.

4. The recombinant polypeptide of claim 1 , wherein the prenylation reaction comprises the production of CBGA from GPP and Olivetolate or cannabigerovarinic acid (CBGVA) from geranylpyrophosphate (GPP) and divirinic acid or CBGXA from GPP and a 2,4-dihydroxy benzoic acid or 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.

5. A composition comprising a recombinant pathway comprising a polypeptide of claim 1 and a plurality of enzymes that convert glucose to Geranylpyrophosphate (GPP).

6. The composition of claim 5 further comprising a pyruvate dehydrogenase bypass enzymatic pathway comprising a pyruvate oxidase and an acetyl phosphate transferase.

7. The composition of claim 4 , wherein the pathway comprises a purge valve that recycles NADH/NAD.

8. The composition of claim 5 , wherein the pathway comprises 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);

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

(xxii) a mutant aromatic prenyltransferase.

9. The composition of claim 5 , wherein the pathway is supplemented with ATP and olivetolate and the pathway produces a cannabinoid precursor.

10. The composition of claim 9 , wherein the pathway further comprises a cannabidiolic acid synthase.

11. The composition of claim 10 , wherein the pathway produces cannabidiolic acid.

12. A recombinant pathway composition comprising the polypeptide of claim 1 and a plurality of enzymes that convert (iso)prenol to geranylpyrophosphate (GPP).