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Recombinant production of steviol glycosides

a technology of steviol glycosides and recombinant production, which is applied in the direction of transferases, baking gum, bakery products, etc., can solve the problems of undesirable off-flavors of contaminants

Inactive Publication Date: 2016-09-01
CONAGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a technology for producing steviol glycosides, which are used as sweeteners. The technology involves using recombinant polypeptides to glycosylate stevioside and rebaudioside compounds. The recombinant polypeptides have been found to have high activity and specificity towards certain steviol glycosides. The technology also includes a method for synthesizing rebaudioside Z, a new steviol glycoside compound. Overall, the technology allows for the efficient and cost-effective production of steviol glycosides.

Problems solved by technology

In particular, variations of the extraction conditions can lead to inconsistent compositions of the steviol glycosides in the stevia extracts, such that the taste profile varies among different batches of extraction products.
These contaminants typically have off-flavors undesirable for the use of the stevia extract as a sweetener.

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

example 1

Selection of Candidate UGT Genes

[0135]Phylogenetic and protein BLAST analysis were used to identify 7 candidate genes belonging to the UGT91 subfamily for 1,2-19-O-glucose glycosylation activity (Table 1).

TABLE 1List of UGT candidate genesNameDescriptionAccessionSequence IDBD1PREDICTED: UDP-XP_003560664.1SEQ ID NO: 1glycosyltransferase 91C1-like[Brachypodium distachyon]BD2PREDICTED: UDP-XP_003560669.1SEQ ID NO: 2glycosyltransferase 91C1-like[Brachypodium distachyon]BD3PREDICTED: LOWXP_003581636.1SEQ ID NO: 3QUALITY PROTEIN: UDP-glycosyltransferase 91C1-like[Brachypodium distachyon]BD4PREDICTED: UDP-XP_003580515.1SEQ ID NO: 4glycosyltransferase 91C1-like[Brachypodium distachyon]BD5PREDICTED: LOWXP_003559500.1SEQ ID NO: 5QUALITY PROTEIN: UDP-glycosyltransferase 91B1-like[Brachypodium distachyon]HV1predicted protein [HordeumBAJ98242.1SEQ ID NO: 6vulgare subsp. vulgare]HV2predicted protein [HordeumBAJ93155.1SEQ ID NO: 8vulgare subsp. vulgare]

example 2

Enzymatic Activity Screening of Candidate UGT Genes

[0136]Full length DNA fragments of all candidate UGT genes were commercially synthesized. Almost all codons of the cDNA were changed to those preferred for E. coli (Genscript, NJ). The synthesized DNA was cloned into a bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0137]Each expression construct was transformed into E. coli BL21 (DE3), which was subsequently grown in LB media containing 50 μg / mL kanamycin at 37° C. until reaching an OD600 of 0.8-1.0. Protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was further grown at 16° C. for 22 hr. Cells were harvested by centrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collected and were either used immediately or stored at −80° C.

[0138]The cell pellets typically were re-suspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 ug / ml lysozyme, 5 ug / ml DNase I, 20 mM imidazole, 500 mM...

example 3

Steviol Glycoside Biosynthesis Using the Recombinant HV1 Polypeptide

[0146]As shown in FIGS. 1A-1C, rebaudioside D can also be formed by glycosylation of the C-3′ of the C-13-O-glucose of rebaudioside E. Thus, rebaudioside D can be produced by different biosynthetic routes (e.g. via rebaudioside A vs. rebaudioside E), depending on the orders in which the glycosylation reactions occur. For example, glycosylation at C-3′ of the C-13-O-glucose of stevioside can occur first to produce the intermediate rebaudioside A, followed by glycosylation at C-2′ of the 19-O-glucose of rebaudioside A to produce rebaudioside D. So far, UGT76G1 (SEQ ID NO:11) from stevia has been identified as an enzyme that transfers a sugar residue to C-3′ of the C-13-O-glucose of stevioside to form rebaudioside A.

[0147]Codon optimized UGT76G1 cDNA was inserted in a bacterial expression vector, and the recombinant UGT76G1 protein was expressed and purified by affinity chromatography. The purified recombinant UGT76G1 ...

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Abstract

Recombinant polypeptides having UDP-glycosyltransferase activities, including a 1,2-19-O-glucose glycosylation activity and a 1,2-13-O-glucose glycosylation activity for synthesizing of steviol glucosides, are provided. A method of producing a steviol glycoside composition using such recombinant polypeptide is also provided. Also disclosed are steviol glycosides referred to as rebaudioside Z1 and rebaudioside Z2.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application No. 61 / 898,571, filed on Nov. 1, 2013, which is hereby incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING[0002]A paper copy of the Sequence Listing and a computer readable form of the sequence containing the file named 32559-17_ST25.txt, which is 46,751 bytes in size (as measured in Microsoft WINDOWS® Explorer), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-12.BACKGROUND[0003]The present disclosure relates generally to the biosynthesis of steviol glycosides. In particular, the present disclosure relates to a recombinant polypeptide that catalyzes the production of steviol glycosides such as rebaudioside D, rebaudioside E and a novel rebaudioside (rebaudioside Z).[0004]Steviol glycosides are natural products isolated from Stevia rebaudiana leaves, and are widely used as high intens...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/10C12P19/56
CPCC12N9/1048A23L1/2363C12Y204/00C12P19/56A23V2002/00A23L1/3002A23G3/38A23G3/36A23G4/06A23L27/36A23L27/33A23L33/105C12N9/1051C12N9/1062C12N15/52C12Y204/01C07H15/24A21D13/062A23L2/60
Inventor MAO, GUOHONGYU, XIAODAN
Owner CONAGEN INC
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