Xylosylated steviol glycosides and enzymatic methods for production
AssigneeCargill, Incorporated
InventorsErin EVANS, Daniel Scott GASPARD, Panagiota KYRIAKOU, Erin Kathleen MARASCO, Guo-Hua ZHENG
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Abstract
Disclosed are methods and compositions for producing xylosylated steviol glycosides that include a xylose residue, or an oligosaccharide moiety including a xylose residue, attached to the 19 carbon of the steviol base. A glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity (such as one of SEQ ID NO:1 or a homolog or variant thereof) forms the xylosylated steviol glycoside using a steviol glycoside acceptor having a glucose residue attached to the 19 carbon of the steviol base. Glycosyltransferase variants with increased activity are also described.
Claims33
A method for forming a glycosylated steviol glycoside, the method comprising
- forming a compound of Formula II from a compound of Formula I in vitro or in an engineered cell using a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, wherein
- Formula I is:
- wherein R1 comprises a glucose residue, and R2 comprises one or more sugar residues or is a hydrogen, and (ii) an activated xylose;
- the glycosyltransferase transfers xylose from an activated xylose to the compound of Formula I;
- and
- Formula II is:
- wherein R3 comprises one or more xylose residue(s) added by the glycosyltransferase, and R4 is the same as R2 or comprises one or more additional sugar residue(s).
An engineered cell comprising
- a pathway for forming a steviol glycoside compound of Formula I:
- wherein R1 comprises a glucose residue, and R2 comprises one or more sugar residues or is hydrogen,
- wherein the pathway provides the steviol glycoside compound of Formula I in an amount that is greater than an amount of steviol glycoside not of formula I that may be formed in the cell;
- and
- a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, wherein the glycosyltransferase is capable of transferring one or more of xylose residues to the compound of Formula I to form a glycosylated steviol glycoside compound of Formula II:
- wherein R3 of Formula II comprises one or more xylose residue(s), and wherein R4 is the same as R2 or includes one or more additional sugar residue(s).
(canceled)
A method for forming a xylosylated steviol glycoside, the method comprising
- forming a xylosylated steviol glycoside product from a steviol glycoside reactant in a reaction composition comprising (i) one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A;
- (ii) UDP-xylose;
- (iii) a polypeptide of SEQ ID NO:1 or SEQ ID NO:2, (iv) a non-activated sugar, and (v) a magnesium and/or manganese salt;
- wherein the reaction composition has a pH in the range of 4.8-7.8;
- wherein the one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A are present at a total of 80% (mol) or greater of all of steviol glycoside in the reaction composition;
- wherein the polypeptide (wt) to the one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A (mol) ratio is in the range of 15 mg:
- 1 mmol to 7.5 mg:
- 1 mmol;
- reacting the reaction composition for at least 12 hours at a temperature in the range of 25-95° C., to provide a product composition comprising one or more of Compounds D, E, G, H, I, or J:
32-37. (canceled)
Dependencies are inferred from the claim text.