Biosynthetic production of variant steviol glycosides
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.
1 . 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:
2 . The method of claim 1 , wherein the sucrose synthase or sucrose synthase domain is selected from the group consisting of an Arabidopsis sucrose synthase I, an Arabidopsis sucrose synthase 3 and a Vigna radiate sucrose synthase.
3 . The method of claim 1 , wherein the UDP-glycosyltransferase comprises the amino acid sequence of SEQ ID NOs: 1 or 3.
4 . The method of claim 1 , further comprising producing rebaudioside D3 by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from the group consisting of sucrose, UDP, UDP-glucose, and combinations thereof.
5 . 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:
6 . The method of claim 5 , wherein the sucrose synthase or sucrose synthase domain is selected from the group consisting of an Arabidopsis sucrose synthase I, an Arabidopsis sucrose synthase 3 and a Vigna radiate sucrose synthase.
7 . The method of claim 5 , wherein the UDP-glycosyltransferase comprises the amino acid sequence of SEQ ID NO: 3.
8 . The method of claim 5 , wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO: 7.
9 . The method of claim 5 , further comprising producing rebaudioside D by incubating rebaudioside E and/or rebaudioside A with a UDP-glycosyltransferase and a substrate selected from the group consisting of sucrose, UDP, UDP-glucose, and combinations thereof.
10 . The method of claim 1 , wherein the reaction mixture is in vitro.
11 . The method of claim 1 , wherein the reaction mixture is a cell-based reaction mixture.