Recombinant production of steviol glycosides

A technology of recombinant gene and host, applied in the production of bulk chemicals, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as odor

Active Publication Date: 2014-04-16
EVOLVA SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stevia extract may also contain impurities such as plant-derived compounds that cause off-flavors
These odors can be more or less problematic depending on food system or application selection

Method used

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  • Recombinant production of steviol glycosides
  • Recombinant production of steviol glycosides
  • Recombinant production of steviol glycosides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0656] Example 1 - Identification of EUGT11

[0657] 15 genes were tested for RebA1,2-glycosylation activity. See Table 10.

[0658] Table 10

[0659] name

source

GenBank accession number

EUGT2

Rice UGT91 homologue

AP003270

EUGT3

Rice UGT91 homologue

AP005171

EUGT4

Rice UGT91 homologue

AP005643

EUGT6

Rice UGT91 homologue

AP005259

EUGT7

Rice UGT91 homologue

AP005171

EUGT8

Rice UGT91 homologue

XM_470006

EUGT9

Rice UGT91 homologue

AP005643

EUGT10

Rice UGT91 homologue

AC133334

EUGT11

Rice UGT91 homologue

AC133334

EUGT12

Rice UGT91 homologue

AC133334

EUGT15

Petunia x hybrid UGT79 homologue

Z25802

EUGT16

Arabidopsis UGT79 homologue

AC004786

[0660] EUGT17

Dianthus caryophyllus UGT79 homologue

AB294391

EUGT18

Morning glory UGT79 homologue

AB192314

...

Embodiment 2

[0663] Identification of embodiment 2-EUGT11 reaction

[0664] EUGT11 was produced by in vitro transcription and translation and incubated with various substrates in the RebD pathway. Similar experiments were performed using in vitro transcribed and translated UGT91D2e. image 3 A schematic diagram showing the 19-O-1,2-diglycosylation reaction by EUGT11 and UGT91D2e. Compounds 1 to 3 were individually identified by mass and expected residence time. image 3 The numbers shown in are the average peak heights of the steviol glycosides obtained from the LC-MS chromatograms and, although not quantitative, can be used to compare the activities of the two enzymes. EUGT11 and UGT91D2e cannot use steviol as a substrate. Both enzymes were able to convert steviol 19-O-monoglycoside (SMG) to the compound, with EUGT11 being approximately 10-fold more efficient than UGT91D2e in converting 19-SMG to compound 1.

[0665] Both enzymes were able to convert rubusoside to stevioside with comp...

Embodiment 4

[0669] Example 4 - Activity of UGT on 19-O-1,2-diglycosylated steviol glycosides

[0670] 19-O-1,2-diglycosylated steviol glycosides produced by EUGT11 require further glycosylation for conversion to RebD. The following experiments were performed to determine whether other UGTs use these intermediates as substrates.

[0671] In one experiment, compound 1 was produced in vitro from 19-SMG by EUGT11 or UGT91D2e in the presence of UDP-glucose. After boiling the samples, UGT85C2 and UDP-glucose were added. Samples were analyzed by LC-MS and compound 2 was detected. This experiment shows that UGT85C2 can use compound 1 as a substrate.

[0672] In another experiment, compound 2 was incubated with UGT91D2e and UDP-glucose. The reaction was analyzed by LC-MS. UGT91D2e cannot convert compound 2 to compound 3 (RebE). Incubation of compound 2 with EUGT11 and UDP-glucose resulted in compound 3. UGT76G1 is able to use RebE as a substrate to produce RebD.

[0673] This suggests that...

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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.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to: U.S. Application Serial No. 61 / 521,084, filed August 8, 2011; U.S. Application Serial No. 61 / 521,203, filed August 8, 2011; Application Serial No. 61 / 521,051; U.S. Application Serial No. 61 / 523,487 filed August 15, 2011; U.S. Application Serial No. 61 / 567,929 filed December 7, 2011 and U.S. Application Serial No. filed February 27, 2012 No. 61 / 603,639, which is hereby incorporated by reference in its entirety. technical field [0003] The present disclosure relates to the recombinant production of steviol glycosides. In particular, the present disclosure relates to the production of steviol glycosides such as rebaudioside D by recombinant hosts such as recombinant microorganisms, plants or plant cells. The present disclosure also provides compositions comprising steviol glycosides. The present disclosure also relates to tools and methods for producing terpenoids by modulating the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/81C12N9/10C12N9/00C12P15/00C12N9/90
CPCC12N15/81C12P15/00C12N9/1051C12N9/1085C12N9/90C12N9/0071C12N15/8243C12P19/44C12N9/88C12N9/0032C12N9/0073C12N15/63C12P19/56Y02P20/52
Inventor 延斯·霍顿-拉森保拉·M·希克斯迈克尔·内斯比通格·托马斯·厄斯特高约恩·汉森迈克尔·达尔格德米克尔森汉森·埃斯本·哈尔克耶尔埃内斯托·西蒙萨比娜·德安德雷德佩雷拉塔瓦雷斯
Owner EVOLVA SA
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