Recombinant production of steviol glycosides

Patent metadata

Field	Value
Publication number	US-10435730-B2
Application number	US-201715439660-A
Country	US
Kind code	B2
Filing date	Feb 22, 2017
Priority date	Aug 8, 2011
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

First claim

The invention claimed is:

1. A method for producing a target steviol glycoside or a target steviol glycoside composition, comprising contacting a starting composition comprising a steviol, a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose, and/or a mixture thereof with a first polypeptide capable of beta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152; and one or more of:
(a) a second polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:5;
(b) a polypeptide capable of rhamnosylation of a steviol 13-O-monoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:150;
(c) a polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT74 family, comprising a UGT74G1 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-19 carboxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;
(d) a polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT85 family, comprising a UGT85C2 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-13 hydroxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; and/or
(e) a polypeptide capable of beta 1,3 glycosylation of a C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
wherein at least one of the polypeptides is a recombinant polypeptide;
and one or more UDP-sugars, under suitable reaction conditions for the transfer of one or more sugar moieties from the one or more UDP-sugars to the steviol, the precursor steviol glycoside, and/or the mixture thereof,
thereby producing the target steviol glycoside or the target steviol glycoside composition.

2. The method of claim 1 , wherein the one or more UDP-sugar comprises UDP-glucose, UDP-rhamnose, fructose, and/or UDP-xylose.

3. The method of claim 1 , wherein the target steviol glycoside comprises steviol-1,2-bioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, or dulcoside A.

4. The method of claim 1 , wherein the target steviol glycoside composition comprises steviol-1,2-bioside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A and a combination thereof.

5. The method of claim 1 , wherein:
(a) the precursor steviol glycoside is steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, stevioside, or rebaudioside B and/or a mixture thereof, the one or more UDP-sugar is UDP-glucose,
the steviol, the precursor steviol glycoside and/or the mixture thereof is contacted with the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152 and one or more of: the polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT85 family, comprising the UGT85C2 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-13 hydroxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, the polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT74 family, comprising the UGT74G1 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-19 carboxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; and the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and
rebaudioside A is produced upon transfer of the one or more sugar moieties from the one or more UDP-glucose to steviol, the precursor steviol glycoside, and/or the mixture thereof; or

(b) the precursor steviol glycoside is steviol-13-O-glucoside or steviol-1,2-bioside and/or a mixture thereof, the one or more UDP-sugar is UDP-glucose,
the steviol, the precursor steviol glycoside and/or the mixture thereof is contacted with the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152 and one or more of: the polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT85 family, comprising the UGT85C2 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-13 hydroxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:5; and the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and
rebaudioside B is produced upon transfer of the one or more sugar moieties from the one or more UDP-glucose to steviol, the precursor steviol glycoside, and/or the mixture thereof; or

(c) the precursor steviol glycoside is steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, or stevioside and/or a mixture thereof, the one or more UDP-sugar is UDP-glucose,
the steviol, the precursor steviol glycoside and/or the mixture thereof is contacted with the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152 and one or more of: the polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT85 family, comprising the UGT85C2 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-13 hydroxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, the polypeptide of uridine 5′-diphospho (UDP) glycosyl transferase (UGT) UGT74 family, comprising the UGT74G1 polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-19 carboxyl group and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; the polypeptide capable of beta 1,2 glycosylat

Assignees

• Evolva Sa

Inventors

• Houghton-Larsen Jens
• Hicks Paula
• Naesby Michael
• Tange Ostergaard Thomas
• Hansen Jorgen
• Mikkelsen Dalgaard Michael
• Hansen Halkjaer Esben
• Simon Ernesto
• Tavares Sabina De Andrade Periera

Classifications

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12P19/56 Primary

  having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin · CPC title