Methods and materials for enzymatic synthesis of mogroside compounds

Patent metadata

Field	Value
Publication number	US-10738340-B2
Application number	US-201815889743-A
Country	US
Kind code	B2
Filing date	Feb 6, 2018
Priority date	Nov 23, 2011
Publication date	Aug 11, 2020
Grant date	Aug 11, 2020

Abstract

Methods and materials for enzymatic synthesis of mogroside compounds described.

First claim

The invention claimed is:

1. An in vitro method for transferring a sugar moiety to a mogrol, a glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound, comprising contacting the mogrol, the glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound with one or more recombinant polypeptides capable of glycosylation at C3′-OH, C24′-OH, C3′-OH and C24′-OH, or C25′-OH of the mogrol, at C3′-OH or C24′-OH of the glycosylated mogroside compound, or both C3′-OH, C24′-OH, C3′-OH and C24′-OH, or C25′-OH of the mogrol and C3′-OH or C24′-OH of the glycosylated mogroside compound, 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 mogrol, the glycosylated mogroside compound, or both the mogrol and the glycosylated mogroside compound;
wherein a mogroside compound glycosylated at C3′-OH, a mogroside compound glycosylated at C24′-OH, a mogroside compound glycosylated at C25′-OH, a mogroside compound glycosylated at C3′-OH and C24′-OH, a mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position, an isomer thereof, and/or a mogroside composition thereof is produced upon transfer of the sugar moiety; and
wherein the one or more recombinant polypeptides comprise at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs. 1-5.

2. The method of claim 1 , wherein:
(a) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C3′-OH is produced upon transfer of the glucose moiety to C3′-OH of the mogrol;
(b) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C24′-OH is produced upon transfer of the glucose moiety to C24′-OH of the mogrol;
(c) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C25′-OH is produced upon transfer of the glucose moiety to C25′-OH of the mogrol;
(d) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C3′-OH and C24′-OH of the mogrol;
(e) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C3′-OH of the mogroside compound glycosylated at C24′-OH;
(f) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C24′-OH of the mogroside compound glycosylated at C3′-OH; and
(g) the UDP-sugar is UDP-glucose, and the mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position is produced upon transfer of the glucose moiety to C3′-OH of the mogroside compound tri-glycosylated at C24′ position.

3. The method of claim 1 , wherein:
(a) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and the mogroside compound glycosylated at C3′-OH is produced upon transfer of the glucose moiety to C3′-OH of the mogrol;
(b) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-5, and the mogroside compound glycosylated at C24′-OH is produced upon transfer of the glucose moiety to C24′-OH of the mogrol;
(c) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4, and the mogroside compound glycosylated at C25′-OH is produced upon transfer of the glucose moiety to C25′-OH of the mogrol;
(d) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-5, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C3′-OH and C24′-OH of the mogrol;
(e) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1 or 3-5, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C3′-OH of the mogroside compound glycosylated at C24′-OH;
(f) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety to C24′-OH of the mogroside compound glycosylated at C3′-OH; and
(g) the UDP-sugar is UDP-glucose, the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and the mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position is produced upon transfer of the glucose moiety to C3′-OH of the mogroside compound tri-glycosylated at C24′ position.

4. The method of claim 1 , wherein:
(a) the mogroside compound glycosylated at C3′-OH is produced upon transfer of the glucose moiety of the UDP-glucose to C3′-OH of the mogrol effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
(b) the mogroside compound glycosylated at C24′-OH is produced upon transfer of the glucose moiety of the UDP-glucose C24′-OH of the mogrol effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-5;
(c) the mogroside compound glycosylated at C25′-OH is produced upon transfer of the glucose moiety of the UDP-glucose to C25′-OH of the mogrol effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;
(d) the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety of the UDP-glucose to C3′-OH and C24′-OH of the mogrol effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1-5;
(e) the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety of the UDP-glucose to C3′-OH of the mogroside compound glycosylated at C24′-OH effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:1 or 3-5;
(f) the mogroside compound glycosylated at C3′-OH and C24′-OH is produced upon transfer of the glucose moiety of the UDP-glucose to C24′-OH of the mogroside compound glycosylated at C3′-OH effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and
(g) the mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position is produced upon transfer of the glucose moiety of the UDP-glucose to C3′-OH of the mogroside compound tri-glycosylated at C24′ position effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

5. The method of claim 1 , further comprising isolating the mogroside compound glycosylated at C3′-OH, the mogroside compound glycosylated at C24′-OH, the mogroside compound glycosylated at C25′-OH, the mogroside compound glycosylated at C3′-OH and C24′-OH, the mogroside compound glycosylated at C3′-OH and tri-glycosylated at C24′ position, the isomer the

Assignees

• Evolva Sa

Inventors

• Liu Yaoquan
• Lee Jung Yeop
• Khare Monika

Classifications

• C12P19/18 Primary

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12P33/20 Primary

  containing heterocyclic rings · CPC title

• C12P33/00

  Preparation of steroids · CPC title