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Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application of recombinant saccharomyces cerevisiae

A technology of gastrodin and glycosyltransferase, which is applied in the field of genetic engineering and can solve the problems of scarcity of glycosyltransferases, synthesis of gastrosyltransferase, etc.

Pending Publication Date: 2021-12-07
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glycosyltransferases currently used for the synthesis of gastrodin are rare, and there is no report on the synthesis of gastrodin in yeast

Method used

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  • Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application of recombinant saccharomyces cerevisiae
  • Recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application of recombinant saccharomyces cerevisiae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1. Verification of the function of glycosyltransferase AsUGT catalyzing the synthesis of gastrodin

[0115] The pCf301 and pCf302 plasmids are self-constructed vectors, which are pESC-derived plasmids with 2μm ori. These two plasmids are recorded in the document "Jiang, J.J., et al. (2018). Metabolic Engineering of Saccharomycescerevisiae for High-Level Production of Salidroside from Glucose. Journal of Agricultural and Food Chemistry 66 (17): 4431-4438", public It can be obtained from the applicant, and can only be used for repeating the experiment of the present invention, and cannot be used for other purposes.

[0116] AsUGT shown in SEQ ID No.3 syn The gene was cloned between the NheI / and AatII restriction sites of the pCf301 vector, and the correct recombinant plasmid obtained after sequencing verification was named pCf301-AsUGT syn .

[0117] Transformation plasmid pCf301-AsUGT syn To Saccharomyces cerevisiae BY4742 (preserved in this laboratory, this...

Embodiment 2

[0120] Example 2, Saccharomyces cerevisiae gastrodin synthesis strain 4742-pGS, Δ7-pGS construction

[0121] gene CAR syn and PPTcg-1-1 syn They were respectively cloned between the NheI / AatII and HindIII / BamHI restriction sites of the pCf302 plasmid to obtain the plasmid pCf302-CP.

[0122] gene ARO4 K229L , AsUGT syn and ubiC syn They were respectively cloned between the SpeI / BglII, NheI / AatII and HindIII / BamHI restriction sites of pCf301 to obtain the plasmid pCf301-AUA.

[0123] Saccharomyces cerevisiae BY4742 gene ARO7 knockout strain Δaro7 is a strain obtained by knocking out the chorismate mutase ARO7 gene on the genome of Saccharomyces cerevisiae BY4742 through conventional yeast homologous recombination gene knockout technology based on pUG6 and pSH65 plasmids. The specific construction method of Δaro7 is as follows: first, use ARO7-UP-P1 and ARO7-DN-P2 as primers, and use the 1.6kb fragment recovered after NotI digestion of pUG6 plasmid as a template to perform ...

Embodiment 3

[0127] Embodiment 3, Saccharomyces cerevisiae gastrodin synthetic strain rGS3 construction

[0128] ARO4 K229L and gastrodin synthesis pathway genes including CAR syn , PPTcg-1 syn , AsUGT syn and ubiC syn With the URA3 tag, a total of 14kb nucleic acid sequence was integrated at the rDNA position of the Saccharomyces cerevisiae Δaro7 genome.

[0129] The technical method is as follows: the rDNA-up (476bp) and rDNA-down (326bp) were amplified from the Saccharomyces cerevisiae BY4742 genome with PCR primer pairs rU-F&R and rD-F&R, respectively. Using r5.8-F&R primers, the plasmid pCf302-CP was used as a template to amplify the CAR containing syn ,PPTcg-1 syn The large fragment is about 5.8kb. Using r7.4-F&R. as primer and plasmid pCf301-AUA as template to amplify to obtain AsUGT syn , ubiC syn ,ARO4 K229L and the large fragment of URA3 about 7.4 kb. Fragment rDNA-up, 5.8kb fragment, 7.4kb fragment and fragment rDNA-down are adjacent in sequence, and there are 40bp ho...

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Abstract

The invention discloses recombinant saccharomyces cerevisiae for producing gastrodin by using glucose and application of the recombinant saccharomyces cerevisiae. The construction method of the engineering strain comprises the step of enabling recipient bacteria to express UDP-glucosyltransferase derived from hedyotis diffusa. The recombinant saccharomyces cerevisiae does not express an ARO7 gene, contains genes AsUGTsyn, CARsyn, PPTcg-1syn and ubiCsyn capable of expressing the gastrodin synthesis pathway and genes ppsA, tktA, ARO1, ARO2 and ARO4 mutant ARO4K229L genes capable of expressing gastrodin precursor synthesis enhancement. According to the application, a metabolic pathway for synthesizing gastrodin from glucose is constructed in food-grade saccharomyces cerevisiae by introducing new glycosyl transferase, the fermentation yield of gastrodin in a 250mL shake flask reaches 2.1 g / L through a genome integration technology and improvement of a precursor anabolic flow, a foundation is laid for large-scale industrial production, and important economic values and social benefits are achieved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant saccharomyces cerevisiae that uses glucose to produce gastrodin and uses thereof. Background technique [0002] Natural products are widely used as nutritional supplements, dietary preparations and medicines due to their multiple benefits to human health. Gastrodia elata Blume (Gastrodia elata Blume) is a perennial herbaceous symbiotic plant of Orchidaceae. It uses underground tubers as medicine. It is a traditional and precious Chinese medicinal material. , convulsions, epilepsy and convulsions in children. Gastrodin, also known as 4-hydroxymethylphenyl-beta-D-glucopyranoside, is the characteristic component of Gastrodia elata, which has good sedative and hypnotic effects, and is effective for neurasthenia, insomnia, and headache symptoms. Clear relief. In addition, gastrodin has also been proven to improve memory, anti-inflammatory, anti-oxidation and other e...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P17/06C12R1/865
CPCC12N15/81C12N9/1051C12N9/0008C12N9/1241C12N9/88C12N9/1022C12N9/1092C12P17/06C12Y102/99006C12Y207/07003C12Y401/0304C12Y202/01001C12Y402/03005C12Y205/01019
Inventor 刘涛殷华庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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