Production of steviol glycoside in recombinant hosts

1. An in vitro method for producing a steviol glycoside, a glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound, comprising:

(a) adding a polypeptide selectively catalyzing conversion of rubusoside to stevioside and having 95% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:17 or 18 and one or more of:

(i) a polypeptide capable of catalyzing beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside and having 95% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:11;

(ii) a polypeptide catalyzing glycosylation of steviol or the steviol glycoside at its C-13 hydroxyl group and having 95% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and/or

(iii) a polypeptide catalyzing beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside having 95% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:9;

and plant-derived or synthetic steviol or steviol glycosides to a reaction mixture;

wherein at least one of the polypeptides is a recombinant polypeptide; and

(b) synthesizing steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound in the reaction mixture.

2. The method of claim 1 , further comprising:

(c) recovering the steviol glycoside, glycosylated ent-kaurenol compound, and/or a glycosylated ent-kaurenoic acid compound alone or a composition comprising the steviol glycoside, glycosylated ent-kaurenol compound, and/or the glycosylated ent-kaurenoic acid compound from the reaction mixture.

3. The method of claim 1 , wherein:

(a) the polypeptide catalyzing beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least one amino acid substitution at residues 93, 99, 114, 144, 148, 152, 195, 196, 199, 211, 213, 221, 286, 384, 426, 438, or 466 of SEQ ID NO:11; and/or

(b) the polypeptide catalyzing glycosylation of steviol or the steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least one amino acid substitution at residues 21, 48, 49, 84, 86, 87, 91, 92, 95, 122, 334, or 334 of SEQ ID NO:7; and/or

(c) the polypeptide catalyzing beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least one amino acid substitution at residues 23, 26, 55, 146, 257, 283, and 337 of SEQ ID NO:9.

4. The method of claim 3 , wherein:

(a) the first polypeptide catalyzing beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least one amino acid substitution of SEQ ID NO:11 that is P93V, S99I, S114F, T144K, T144L, T144M, A148K, M152T, L195G, L195C, L195S, L195N, L195V, V196P, K199C, L211H, L211M, L211I, L211C, L211T, L213E, S221I, V286C, V286N, V286S, G384W, G384K, G384Y, E426G, E438H, E438M, or A466V; and/or

(b) the polypeptide catalyzing glycosylation of steviol or the steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least one amino acid substitution of SEQ ID NO:7 that is Q21L, Q21T, Q21V, F48S, F48H, F48Y, F48R, F48Q, F48W, F48T, I49V, S84G, S84A, S84T, S84C, S84P, S84N, S84V, P86R, P86G, 187H, I87P, I87M, I87Y, L91K, L91R, L91T, L92F, L92I, L92M, I95K, F122S, L334S, or L334M; and or

(c) the polypeptide catalyzing beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having at least one amino acid substitution of SEQ ID NO:9 that is Q23H, I26W, T146G, H155L, L257G, S253W, T284G, S283N, K337P, or T55K.

5. The method of claim 1 , wherein the reaction mixture comprises:

(a) one or more steviol glycosides, glycosylated ent-kaurenol compounds, and/or a glycosylated ent-kaurenoic acid compounds produced in the reaction mixture;

(b) a UGT polypeptide;

(c) UDP-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or

(d) reaction buffer and/or salts.

6. The method of claim 1 , wherein:

(a) the steviol glycoside comprises steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside M (RebM), a di-glycosylated steviol, a tri-glycosylated steviol, a tetra-glycosylated steviol, a penta-glycosylated steviol, a hexa-glycosylated steviol, a hepta-glycosylated steviol, and/or isomers thereof;

(b) the glycosylated ent-kaurenol compound comprises di-glycosylated ent-kaurenol, tri-glycosylated ent-kaurenol, and/or isomers thereof; and

(c) the glycosylated ent-kaurenoic acid compound comprises di-glycosylated ent-kaurenoic acid, tri-glycosylated ent-kaurenoic acid, and/or isomers thereof.

7. The method of claim 6 , wherein:

(a) the di-glycosylated steviol comprises compound 2.23 of Table 1;

(b) the tri-glycosylated steviol comprises compound 3.1 and/or compound 3.34 of Table 1;

(c) the tetra-glycosylated steviol comprises compound 4.26 and/or compound 4.33 of Table 1;

(d) the penta-glycosylated steviol comprises compound 5.22, compound 5.24, and/or compound 5.25 of Table 1;

(e) the hexa-glycosylated steviol comprises compound 6.1 and/or compound 6.23 of Table 1;

(f) the hepta-glycosylated steviol comprises compound 7.2, compound 7.5, and/or compound 7.13 of Table 1;

(g) the glycosylated ent-kaurenoic acid compound comprises compound KA3.1, compound KA3.2, and/or compound KA2.7 of Table 1; and

(h) the glycosylated ent-kaurenol compound comprises compound KL2.8 and/or compound KL3.1 co-eluted with compound KL3.6 of Table 1.

8. The method of claim 7 , wherein:

(a) compound 4.26 has the structure:

(b) compound 5.22 has the structure:

(c) compound 6.1 has the structure:

(d) compound 7.2 has the structure:

(e) compound 7.5 has the structure:

(f) compound KA3.1 has the structure:

(g) compound KA3.2 has the structure:

and

(h) compound KL3.1 has the structure:

9. The method of claim 6 , wherein:

(a) the tri-glycosylated ent-kaurenoic acid comprises a compound having the structure:

(b) the penta-glycosylated steviol comprises a compound having the structure:

(c) the hexa-glycosylated steviol comprises a compound having the structure:

and

(d) the hepta-glycosylated steviol comprises a compound having the structure: