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US 12,668,826 B2Granted
Biosynthetic production of variant steviol glycosides
AssigneeConagen Inc.
InventorsGuohong Mao, Michael Batten, Yang Luo, Oliver Yu
Life of this patent
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Abstract
The present invention relates to novel steviol glycosides R6-1, R6-2A, R6-2B, R6-4A, R6-4B and R7-2 and the production of these novel steviol glycosides, such as through enzymatic bioconversion. The use of these novel steviol glycosides as sweeteners and in orally consumable products are also provided.
Claims11
A method of producing rebaudioside R6-2A and/or R6-2B, the method comprising:
- (I) preparing a reaction mixture comprising:
- (i) rebaudioside D3;
- (ii) one or more substrates selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof;
- and
- (iii) an enzyme selected from the group consisting of:
- (a) a UDP-glycosyltransferase (UGT);
- (b) a UDP-glycosyltransferase and a sucrose synthase separately added to the reaction mixture;
- and
- (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain;
- and
- (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-2A and/or R6-2B;
- wherein the UDP-glycosyltransferase is at least 90% identical to the amino acid sequence of SEQ ID NO:
- 1 or 3;
- wherein the UDP-glycosyltransferase fusion enzyme is at least 90% identical to the amino acid sequence of SEQ ID NO:
- 5 or 7;
- wherein the rebaudioside D3 has the structure of:
- the rebaudioside R6-2A has the structure of:
- the rebaudioside R6-2B has the structure of:
15 more elements
A method of producing rebaudioside R6-1, the method comprising:
- (I) preparing a reaction mixture comprising:
- (i) rebaudioside D;
- (ii) one or more substrates selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof;
- and
- (iii) an enzyme selected from the group consisting of:
- (a) a UDP-glycosyltransferase (UGT), wherein the UDP-glycosyltransferase is at least 90% identical to the amino acid sequence of SEQ ID NO:
- 3;
- (b) a UDP-glycosyltransferase and a sucrose synthase separately added to the reaction mixture;
- and
- (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain, wherein the UDP-glycosyltransferase fusion enzyme is at least 90% identical to the amino acid sequence of SEQ ID NO:
- 7;
- and
- (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-1;
- wherein the rebaudioside D has the structure of:
- the rebaudioside R6-1 has the structure of:
12 more elements
Dependencies are inferred from the claim text.