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Recombinant production of steviol glycosides

A technology of stevia and recombinant host, which is applied in the direction of sugar derivatives, bulk chemical production, recombinant DNA technology, etc., and can solve problems such as odor

Active Publication Date: 2013-06-26
沃维公司
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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

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

[0223] Example 1--Construction of Kaurene Biosynthetic Pathway Gene

[0224] The nucleotide sequence encoding a truncated baker's yeast HMG CoA reductase was cloned into a yeast high copy episomal plasmid vector such that the coding sequence is operably linked to a promoter that is repressed by the amino acid methionine and transcription is regulated by its regulation. See US Patents 5,460,949 and 5,306,862.

[0225] The nucleotide sequence encoding the GGPPS enzyme shown in Table 1 is modified to adapt to expression in yeast (see SEQ ID NOs: 18-25), and cloned into the E. coli vector so that the coding sequence can be operably linked A yeast promoter that is repressed by the amino acid methionine and that regulates transcription. Also shown in Table 1 is the name of each plasmid ("entry vector") containing the expression cassette. SEQ ID NOs: 26-33 give the nucleotide sequences from the source organism from which the polypeptide was originally identified. Using the unmo...

Embodiment 3

[0255] The construction of embodiment 3-yeast strain

[0256] A yeast strain designated EFSC301 was engineered by replacing the endogenous ERG9 promoter with the copper-inducible CUP1 promoter. Strain EFSC301 is a derivative of EUROSCARF deposited yeast strain BY4742. See Internet uni-frankfurt.de / fb15 / mikro / euroscarf / data / by.html. Transcript levels of the ERG9 gene are very low in standard yeast growth media because of the low copper concentrations in such media. The decrease in ergosterol production in this strain resulted in an increase in the amount of isoprene units available for steviol biosynthesis. The yeast strain was also engineered to genomically integrate the Stevia UGT85C2 and UGT74G1 genes, each under the transcriptional control of the strong constitutive GPD1 promoter. See Table 8. The strain contained one copy each of the Stevia UGT85C2 and UGT74G1 genes integrated into the MUS1241 strain genome.

Embodiment 4

[0257] Example 4--Analysis of Steviol Glycoside Pathway Gene Expression in Yeast

[0258] To examine steviol glycoside biosynthesis in yeast, the expression cassettes of the 36 entry vectors in Tables 1-7 and Example 1 were randomly concatenated in ligation reactions to form artificial yeast chromosomes ("eYACs"). The process is as Figure 5 hint.

[0259] Two different sets of ligation reactions were performed. Connectome A included all the genes in Tables 1-7, but not the bifunctional CDPS-KS gene (Table 4). Connectome B included all the genes in Tables 1-7, but did not contain the single-function CDPS and KS genes (Tables 2-3).

[0260] Prepare 30-200 μg of DNA from each entry vector containing the expression cassette. Gene expression cassettes were released from each vector using the restriction enzyme AscI. The expression cassettes were then randomly concatenated into eYACs by a 3-hour ligation reaction using T4 ligase. Because tandem DNA is very viscous, the succ...

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Abstract

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

Description

[0001] sequence listing [0002] This application contains a Sequence Listing submitted via EFS-Web in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy generated on June 2, 2011 is named 25933WO1.txt and is 483,406 bytes in size. technical field [0003] The present disclosure relates to the recombinant production of steviol and steviol glycosides. In particular, the present disclosure relates to the production of steviol and steviol glycosides, such as rubusoside and / or rebaudioside A, by recombinant hosts such as recombinant microorganisms, plants or plant cells. The present disclosure also provides compositions containing the steviol glycosides. Background technique [0004] Sweeteners are well-known ingredients commonly used in the food, beverage or confectionery industry. Sweeteners can be used to add to final foods during production, or used on their own in appropriate dilutions as a tabletop sweetener or as a household substit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N5/04C12P7/58C07H21/04C12N9/10C12P21/04C12N5/00C12N15/00A23L1/236A23L27/30
CPCC12N9/1051C12P19/56C12Q1/6895C12Y204/01126C12Q2600/156C12N15/8243C12P15/00A23L27/36C12N15/52A23L2/60C07H15/256C12N9/0006C12N9/0042C12N9/0073C12N9/1085C12N9/88C12N9/90C12Y101/01088C12Y106/02004C12Y114/13078C12Y114/13079C12Y205/01029C12Y402/03019C12Y505/01012C07H21/04C12P7/58C12Q1/6827C12N15/81C12N15/8245C12P7/42C12N9/1077Y02P20/52
Inventor G.M.基肖尔M.莫申P.M.希克斯J.汉森J.霍顿-拉森E.H.汉森M.D.米克尔森S.塔瓦雷斯C.布洛姆
Owner 沃维公司
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