Biosynthetic production of steviol glycosides and processes therefore

Patent metadata

Field	Value
Publication number	US-11680281-B2
Application number	US-202016904797-A
Country	US
Kind code	B2
Filing date	Jun 18, 2020
Priority date	Oct 14, 2016
Publication date	Jun 20, 2023
Grant date	Jun 20, 2023

Abstract

The present invention relates to the production of steviol glycoside rebaudiosides D4, WB1 and WB2 and the production of rebaudioside M from Reb D4.

First claim

The invention claimed is:

1. A method of producing rebaudioside M, the method comprising:
providing a reaction mixture comprising:
(a) rebaudioside D4 having the structure:

(b) uridine diphosphate-glucose (UDP-glucose); and
(c) a UDP-glycosyltransferase (UGT) comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 3; and

incubating the reaction mixture for a sufficient time to produce rebaudioside M.

2. The method of claim 1 , wherein the UDP-glycosyltransferase is UGT76G1 having the amino acid sequence of SEQ ID NO: 1.

3. The method of claim 1 , wherein the UDP-glycosyltransferase comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 3.

4. The method of claim 1 , wherein the UDP-glycosyltransferase is a mutant of an enzyme having the amino acid sequence of SEQ ID NO: 3, wherein the mutant comprises at least one mutation at an amino acid residue position corresponding to a position selected from 3, 6, 90, 91, 93, 181, 183, 184, 185, 350, 389, 410, 418, 450, 451, 452, and 454 of SEQ ID NO: 3.

5. The method of claim 4 , wherein the UDP-glycosyltransferase is an enzyme having the amino acid sequence of SEQ ID NO: 3 and further having at least one substitution selected from the group consisting of (i) W3L, (ii) L6A or L6G, (iii) T90A or T90G, (iv) S91G or S91L, (v) V93A or V93G, (vi) S181G, (vii) F183V, F183A, or F183G, (viii) G184A, (ix) L185A, (x) G350A, (xi) L389V, (xii) S410G, (xiii) H418V, (xiv) T450A or T450G, (xv) K451A, (xvi) D452A, and (xvii) K454L or K454V.

6. The method of claim 1 , further comprising incubating rebaudioside WB1 having the structure:

with an HV1 UGT enzyme for a sufficient time to produce rebaudioside D4.

7. The method of claim 1 , wherein the reaction mixture further comprises sucrose, uridine diphosphate, and a sucrose synthase.

8. The method of claim 1 , further comprising rebaudioside M thus produced from the reaction mixture for use as a sweetener.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Vick Jacob Edward
• Batten Michael
• Byun David
• Luo Yang
• Wu Yilin
• Zhang Beihua
• Yu Xiaodan

Classifications

• C07H15/256 Primary

  Polyterpene radicals · CPC title

• C12Y204/01013

  Sucrose synthase (2.4.1.13) · CPC title

• C12N15/81

  for yeasts · CPC title

• A23L27/36

  Terpene glycosides · CPC title

• C12Y302/01021

  Beta-glucosidase (3.2.1.21) · CPC title