Engineered Glycosyltransferase, Engineered Polynucleotide, Vector, Host Cell, Method for Producing at Least One Engineered Glycosyltransferase, Composition, and Method

Abstract

The present invention provides engineered glycosyltransferase (gt) enzymes, polypeptides having gt activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides engineered sucrose synthase (sus) enzymes, polypeptides having sus activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the gt enzymes and methods of using the engineered gt enzymes to manufacture products with β-glucose linkages. The present invention further provides compositions and methods for the production of rebaudiosides (e.g., rebaudioside m, rebaudioside a, rebaudioside ie, rebaudioside d). The present invention also provides compositions comprising the enzymes and methods of using them.Methods for producing the gt and sus enzymes are also provided.

Claims

1. Engineered glycosyltransferase, characterized in that it comprises a polypeptide sequence selected from SEQ ID NOS: 4, 8, 32, 232, 348, 548, 562, 696, 4684, 4838, 4876, 5066, 5290, 5372, 5562, 5708, 5976, 6138, 6288, 6468, 6864, 7388, and / or 8088, and wherein said polypeptide sequence of said engineered glycosyltransferase comprises at least one mutation or set of mutations at one or more positions selected from: (a) 309, wherein said position is numbered with reference to SEQ ID NO: 4; or (b) 308, wherein the said position is numbered with reference to the SEQ ID Nos: 696, 4876, 5290 or 5562.

2. Engineered glycosyltransferase, according to claim 1, characterized in that said engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from: (a) 309R, 10- / 309R, 309L, 309N or 309S, wherein the positions are numbered with reference to SEQ ID NO: 4; (b) V309R, R10- / V309R, V309L, V309N or V309S, wherein the positions are numbered with reference to SEQ ID NO: 4; or (c) 309 or 10 / 309, wherein the positions are numbered with reference to SEQ ID NO:

4.

3. Engineered glycosyltransferase, according to claim 1, characterized in that said engineered glycosyltransferase polypeptide sequence comprises at least one mutation or set of mutations at one or more positions selected from: (a) 308F or 308Y, wherein said position is numbered with reference to SEQ ID NO: 696, optionally wherein at least one mutation or set of mutations at one or more positions is selected from R308F or R308Y, wherein said position is numbered with reference to SEQ ID NO: 696; (b) 308C, 308L or 308T, wherein the said position is numbered with reference to SEQ ID NO: 4876, optionally wherein at least one mutation or set of mutations in one or more positions is selected from R308C, R308L or R308T, wherein the said position is numbered with reference to SEQ ID NO: 4876;(c) 308L, wherein said position is numbered with reference to SEQ ID NO: 5290, optionally wherein at least one mutation or set of mutations in one or more positions is selected from R308L, wherein said position is numbered with reference to SEQ ID NO: 5290; (d) 308L or 308Q, wherein said position is numbered with reference to SEQ ID NO: 5562, optionally wherein at least one mutation or set of mutations in one or more positions is selected from R308L or R308Q, wherein said position is numbered with reference to SEQ ID NO: 5562; or (e) 308L, wherein said position is numbered with reference to SEQ ID NO: 7388, optionally wherein at least one mutation or set of mutations at one or more positions is selected from R308L, wherein said position is numbered with reference to SEQ ID NO: 7388.; 4. Engineered glycosyltransferase, according to any one of claims 1-3, characterized in that said engineered glycosyltransferase is a beta-1,3-glycosyltransferase.

5. Engineered glycosyltransferase, according to any one of claims 1-4, characterized in that the aforementioned engineered glycosyltransferase is an NDP-glycosyltransferase selected from ADP-glucose-dependent glycosyltransferases (AGTs), CDP-glucose-dependent glycosyltransferases (CGTs), GDP-glucose-dependent glycosyltransferases (GGTs), TDP-glucose-dependent glycosyltransferases (TGTs), and IDP-dependent glucose glycosyltransferases (IGTs).

6. Engineered glycosyltransferase, according to any one of claims 1-5, characterized in that said engineered glycosyltransferase is an ADP-glucose dependent glycosyltransferase.

7. Engineered polynucleotide, characterized in that it encodes at least one engineered glycosyltransferase polypeptide as defined in any one of claims 1-6; wherein the polynucleotide is selected from SEQ ID NO: 3, 7, 31, 231, 347, 547, 561, 695, 4683, 4837, 4875, 5065, 5289, 5371, 5561, 5707, 5975, 6137, 6287, 6467, 6863, 7387 and 8087.

8. Vector, characterized in that it comprises at least one engineered polynucleotide as defined in claim 7, optionally wherein said vector further comprises at least one control sequence; wherein the polynucleotide is selected from the SEQ ID NO: 3, 7, 31, 231, 347, 547, 561, 695, 4683, 4837, 4875, 5065, 5289, 5371, 5561, 5707, 5975, 6137, 6287, 6467, 6863, 7387 and 8087.

9. Microbial host cell, characterized in that it comprises: (a) at least one engineered polynucleotide as defined in claim 7; or (b) at least one vector as defined in claim 8.

10. Microbial host cell, characterized by the fact that said host cell is selected from a eukaryotic and prokaryotic organism. Petition 870260055468, dated 08 / 06 / 2026, page 13 / 26 4 / 8 11. A method for producing at least one engineered glycosyltransferase as defined in any one of claims 1-7, characterized in that it comprises culturing the host cell, as defined in claim 9, under conditions such that said engineered glycosyltransferase is produced by said host cell, optionally wherein the method further comprises the step of recovering said engineered glycosyltransferase.

12. Composition, characterized in that it comprises at least one engineered glycosyltransferase, as defined in any one of claims 1-7.

13. Method, characterized in that it is for: (a) glycosylation of a substrate, comprising providing at least one substrate, at least one engineered glycosyltransferase as defined in any of claims 1-7, and contacting said substrate with said glycosyltransferase under conditions such that said substrate is glycosylated to produce at least one glycosylated product, optionally wherein: (i) said substrate comprises at least one steviol glycoside; or (ii) said glycosylated product comprises at least one monoglycosylated and / or polyglycosylated product; (b) production of rebaudioside M, comprising providing a rebaudioside D substrate, NDP-glucose, and at least one engineered glycosyltransferase as defined in claims 1-7, combining said rebaudioside D substrate, NDP-glucose and said glycosyltransferase under conditions such that rebaudioside M is produced;(c) producing rebaudioside A and / or rebaudioside I, comprising providing a stevioside substrate, NDP-glucose, and at least one engineered glycosyltransferase as defined in claims 1-7, combining said stevioside substrate, NDP-glucose and glycosyltransferase under conditions such that rebaudioside A and / or rebaudioside I is produced; or (d) production of rebaudioside D, comprising providing a stevioside substrate, NDP-glucose, and at least one engineered glycosyltransferase as defined in claims 1-7, combining said stevioside substrate, NDP-glucose and glycosyltransferase under conditions such that rebaudioside D is produced.

14. Method according to claim 13, characterized in that said conditions are in vitro or within a host cell that is cultured ex vivo.

15. Method according to claim 13 or 14, characterized in that: (a) said NDP-glucose is selected from ADP-glucose, CDP-glucose, TDP-glucose, GDP-glucose and / or IDT-glucose; (b) said NDP-glucose is not UDP-glucose; and / or (c) said NDP-glucose is ADP-glucose.

16. Method, characterized in that it is for: (a) the production of rebaudioside M comprising providing a substrate of rebaudioside D, NDP, sucrose, a sucrose synthase and at least one engineered glycosyltransferase as defined in claims 1-7, combining said substrate of rebaudioside D, NDP, sucrose, sucrose synthase and glycosyltransferase under conditions such that rebaudioside M is produced; (b) the production of rebaudioside A and / or rebaudioside I comprising providing a substrate of stevioside, NDP, sucrose, a sucrose synthase and at least one engineered glycosyltransferase as defined in claims 1-7, combining said substrate of Petition 870260055468, dated 08 / 06 / 2026, page. 15 / 26 6 / 8 stevioside substrate, NDP, sucrose, sucrose synthase and glycosyltransferase under conditions such that rebaudioside A and / or rebaudioside I is produced;(c) the production of rebaudioside D, comprising providing a stevioside substrate, NDP, sucrose, a sucrose synthase and at least one engineered glycosyltransferase as defined in claims 1-7, combining said stevioside substrate, NDP, sucrose, sucrose synthase and glycosyltransferase under conditions such that rebaudioside D is produced; (d) the production of rebaudioside M, comprising providing a stevioside substrate comprising at least one stevioside and / or a mixture of stevioside and rebaudioside A, NDP, sucrose, a sucrose synthase and at least one engineered glycosyltransferase as defined in claims 1-7, combining said stevioside substrate, NDP, sucrose, sucrose synthase and glycosyltransferase under conditions such that rebaudioside M is produced;or (e) the production of rebaudioside M, comprising providing a stevioside substrate, NDP, sucrose, at least one sucrose synthase and at least one engineered glycosyltransferase as defined in claims 1-7, combining said stevioside substrate, NDP and glycosyltransferase under conditions such that rebaudioside A is initially produced, rebaudioside D and / or rebaudioside I is then produced and rebaudioside M is finally produced.

17. Method according to claim 16, characterized in that said conditions are in vitro or within a host cell that is cultured ex vivo.

18. Method according to claim 16 or 17, characterized in that said sucrose synthase is an engineered sucrose synthase comprising a polypeptide sequence selected from SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506, or 8420.

19. Method according to any one of claims 13 to 18, characterized in that: (a) said method is conducted as a single-vessel reaction, optionally wherein: (i) said method is conducted sequentially; or (ii) the method also comprises repeating the steps of the methods, for example, wherein: (i) said sucrose is recycled during repeated steps; or (ii) engineered glycosyltransferase and / or other reaction components are recycled; (b) said stevioside substrate is: (i) extracted from Stevia rebaudiana; or (ii) is produced synthetically; (c) said glycosyltransferase and / or said sucrose synthase is immobilized; (d) the method produces a reaction product that includes fructose, optionally wherein said fructose is removed from said reaction product; (e) the method further comprises a washing step; (f) the method further comprises at least one column chromatography step;(g) said at least one engineered glycosyltransferase is a beta-1,3 glycosyltransferase selected from the glycosyltransferase as defined in any of claims 1-7; and / or (h) the method further comprises at least one engineered sucrose synthase, wherein the engineered sucrose synthase comprises Petition 870260055468, dated 08 / 06 / 2026, page 17 / 26 8 / 8 a polypeptide sequence selected from SEQ ID NO: 74, 1080, 1158, 1222, 1392, 1456, 1582, 1764, 1804, 1840, 2064, 2432, 2510, 7506, or 8420.;

Images