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Method for producing stevioside compounds by using microorganisms

A technology of steviol glycosides and compounds, applied in the field of synthetic biology

Active Publication Date: 2014-04-09
SICHUAN INGIA BIOSYNTHETIC CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It will have the advantages of low cost, less floor space, easy control of product quality, etc., but there is no report on heterologous biosynthesis of rebaudioside A. The key technical problem is that in rebaudioside A biological In the synthetic pathway, the transferase from steviol monoglycoside to steviol diglycoside by one-step glycosylation is unknown

Method used

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  • Method for producing stevioside compounds by using microorganisms
  • Method for producing stevioside compounds by using microorganisms
  • Method for producing stevioside compounds by using microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0203] Embodiment 1, Obtaining of proteins used in heterologous synthetic pathways of steviol glycoside compounds

[0204] Geranylgeranyl pyrophosphate synthase, Cuban pyrophosphate synthase, conchicene synthase, bifunctional conchicene synthase, conchicene oxidation Gene origin and synthesis of enzymes, conchiate-13α-hydroxylase, UGT85C2 glycosyltransferase, UGTB1 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase and cytochrome P450 redox proteins.

[0205] First, geranylgeranyl pyrophosphate synthase (GGPPS) from Canadian yew (Taxus canadensis) and stevia (Stevia rebaudiana) was selected from the National Bioinformatics Database (NCBI). Cuban pyrophosphate synthase (CDPS) from Stevia rebaudiana and Bradyrhizobium japonicum, shellene synthase (KS) from Stevia rebaudiana and Bradyrhizobium japonicum ), from Physcomitrella patens and Gibberella fujikuroi bifunctional conchene synthase (CPS / KS), from Stevia rebaudiana, Arabidopsis thaliana, Conchi...

Embodiment 2

[0242] Example 2 , Construction of prokaryotic expression vector

[0243] The geranylgeranyl pyrophosphate synthase, cubanpyrophosphate synthase, conchicene synthase, bifunctional conchicene synthase, conchicene oxidase, conchiolic acid-13α-hydroxy Clase, UGT85C2 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase and cytochrome P450 redox protein related genes were cloned into the corresponding plasmids, and the bacterial rebaudioside A synthesis pathway gene expression vector was cloned. Construct.

[0244] After the stop codon TAA of the optimized ggpps gene, a SpeI restriction site was added, and then it was cloned into the NcoI / HindIII restriction site of plasmid pET28a (purchased from Novagen) to obtain pET28a-ggpps ( image 3 A).

[0245] After the stop codon TAA of 9 genes of optimized cdps, cps / ks, ks, ko, kah, ugt85c2, ugtb1, ugt74g1, ugt76g1, SpeI sites were respectively added, and then these genes were respectively cloned into plasmid ...

Embodiment 3

[0258] Example 3 , Construction of fungal expression vector

[0259] The geranylgeranyl pyrophosphate synthase, cubanpyrophosphate synthase, conchicene synthase, bifunctional conchicene synthase, conchicene oxidase, conchiolic acid-13α-hydroxy Clase, UGT85C2 glycosyltransferase, UGTB1 glycosyltransferase, UGT74G1 glycosyltransferase, UGT76G1 glycosyltransferase and cytochrome P450 redox protein related genes were cloned into the corresponding plasmids, and fungal rebaudioside A Construction of synthetic gene expression plasmids.

[0260]First, the original pAO815 vector (purchased from Invitrogen) was transformed, and BamHI and XhoI restriction sites were introduced after the terminator of pAO815 by site-directed mutagenesis PCR. The transformed pAO815 was named pSY01. The BamHI site in the pET28a-ggpps gene was removed by site-directed mutagenesis PCR. The BglII site in the pET21a-ks gene was removed by site-directed mutagenesis PCR.

[0261] Using pET28a-ggpps as a temp...

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Abstract

The invention relates to a method for producing stevioside compounds by using microorganisms. According to the present invention, the key enzymes for heterologous biosynthesis of the stevioside compounds are disclosed in the first time, wherein the applied enzymes comprise GGPPS, one selected from a CDPS and KS mixture and CPS / KS, KO, CPR, kaurenoic acid-13alpha-hydroxylase, UGT85C2 glycosyltransferase and UGTB1 / IBGT glycosyltransferase, and further optionally comprise UGT74G1 glycosyltransferase and / or UGT76G1 glycosyltransferase so as to carry out heterologous biosynthesis of the stevioside compounds.

Description

technical field [0001] The invention belongs to the field of synthetic biology; more specifically, the invention relates to a method for producing steviol glycosides by using microorganisms. Background technique [0002] Rebaudioside A (RebA) is a new type of natural sweetener extracted from Stevia rebaudiana. It has the characteristics of high sweetness, low calorie content and good stability. Compared with other steviol glycosides, rebaudioside A has the highest sweetness, about 450 times that of sucrose, and its calorific value is only 1 / 300 of that of sucrose. Rebaudioside A has high sweetness, white color, pure sweet taste, and no peculiar smell, so it is the best natural substitute for sucrose and chemically synthesized sweeteners, and is known internationally as "the third sugar source in the world" . [0003] The structural formula of steviol glycosides isolated from Stevia rebaudiana at present is as follows: figure 1 As shown, as R1 and R2 are different, steviol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/56C12R1/645
CPCC12P19/56C12N9/1051C12N15/63C07K14/415C12N9/1048Y02P20/52
Inventor 王勇熊智强李诗渊汪建峰
Owner SICHUAN INGIA BIOSYNTHETIC CO LTD
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