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Recombinant escherichia coli for utilizing glucose to produce p-hydroxybenzyl alcohol or gastrodin and application

A technology of recombinant Escherichia coli and p-hydroxybenzyl alcohol, applied in the field of bioengineering, can solve the problems of low titer, long reaction cycle of plant tissue culture, environmental damage of heavy metal catalysts, etc., and achieve the effect of increasing production

Active Publication Date: 2015-08-19
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

[0009] The chemical synthesis process is relatively mature, but the addition of heavy metal catalysts will cause great damage to the environment; the chemical synthesis method does not require red phosphorus and bromine, and the yield is low; the conversion efficiency of microorganisms is low, and exogenous substrates need to be added ;Plant tissue culture reaction cycle is long, titer is low
So far, there is no report on the de novo synthesis of gastrodin by microorganisms

Method used

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  • Recombinant escherichia coli for utilizing glucose to produce p-hydroxybenzyl alcohol or gastrodin and application
  • Recombinant escherichia coli for utilizing glucose to produce p-hydroxybenzyl alcohol or gastrodin and application
  • Recombinant escherichia coli for utilizing glucose to produce p-hydroxybenzyl alcohol or gastrodin and application

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Experimental program
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Effect test

Embodiment 1

[0053] The Escherichia coli expression vector is pCDFDuet-aroG*-ppsA-pgm-galU, and the preparation method of the vector preferably comprises the following steps:

[0054] (a) With reference to Chinese patent (patent application number: 201410115011.4.), Bi Huiping, Bai Yanfen, etc., a high-yielding E. coli expression strain of tyrosol and / or salidroside and icariside D2 and its application, the plasmid pBbA5c -tyrA*-aroG*-ppsA was used as a template, digested with PstI and AflII, and then ligated into plasmid pCDFDuet-1 digested with PstI and AflII to obtain plasmid pCDFDuet-aroG*-ppsA.

[0055] (b) Using the Escherichia coli BL21 (DE3) genome as a template, with primers pgm-5FPFseI (SEQ ID No: 6) / pgm-3RFPAatII (SEQ ID No: 7) / galU-5FPBamHI (SEQ ID No: 8) / galU -3RPXhoI (SEQ ID No: 9) was amplified by primers to obtain pgm and galU fragments, and the pgm fragment was double-cut with FseI / AatII and ligated into the vector pCDFDuet to obtain the Escherichia coli expression vector ...

Embodiment 2

[0058] Escherichia coli expression vector is pETDuet-ubiC-CAR-Sfp, Escherichia coli expression vector pETDuet-ubiC-CAR-Sfp-ugt73b6, Escherichia coli expression vector pETDuet-ubiC-CAR-Sfp-ugt73b6 FS The gene preparation method preferably comprises the following steps:

[0059] (a) According to literature reports, design a suitable restriction enzyme site, optimize the synthesis of the codon-optimized CAR-Sfp gene from Shanghai Jierui Bioengineering Co., Ltd., and digest the synthetic fragment with EcoRI and PstI, and then ligate it into EcoRI and PstI From the digested plasmid pETDuet, the plasmid pETDuet-CAR-Sfp was constructed.

[0060] (b) Using primer ubiC-5FPNdeI (SEQ ID No: 10) / ubiC-3RPBglII (SEQ ID No: 11) as a primer, using the genomic DNA of Escherichia coli (Escherichia coli) BL21 (DE3) as a template to perform PCR and amplify The ORF of ubiC was obtained, and the amplified product was digested with NdeI and BglII and then ligated into the plasmid pETDuet-CAR-Sfp di...

Embodiment 3

[0093] Reconstitution of recombinant E. coli strains producing p-hydroxybenzyl alcohol using glucose and reconstitution of recombinant E. coli strains producing gastrodin using glucose

[0094] The plasmids pCDFDuet-aroG*-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp were transformed into E. coli strain BL21(DE3) by chemical transformation;

[0095] The plasmids pCDFDuet-aroG*-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6 were transformed into E. coli strain BL21(DE3) by chemical transformation;

[0096] Plasmids pCDFDuet-aroG*-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6 FS Transformed into Escherichia coli strain BL21(DE3) by means of chemical transformation;

[0097] The transformation method is as follows: take 100 μl of competent Escherichia coli strain BL21 (DE3) cells on ice, add 2 μl of plasmid pCDFDuet-aroG*-ppsA-pgm-galU and 2 μl of plasmid pETDuet-ubiC-CAR-Sfp or pETDuet-ubiC-CAR-Sfp-ugt73b6 or pETDuet-ubiC-CAR-Sfp-ugt73b6 FS , mix gently, place on ice for 30 minutes...

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Abstract

The invention discloses recombinant escherichia coli for utilizing glucose to produce p-hydroxybenzyl alcohol or gastrodin and application. The recombinant escherichia coli for utilizing glucose to produce the gastrodin comprises two expression vectors of pCDFDuet-aroG*-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6FS; the aro-G* gene is represented by SEQ ID NO:1. According to the invention, by constructing a new p-hydroxybenzyl alcohol synthetic pathway and regulating and controlling metabolic flux from glucose to tyrosine, the escherichia coli with high yield of gastrodigenin and p-hydroxybenzyl alcohol is obtained; yield of p-hydroxybenzyl alcohol reaches 240mg / L; a high-efficiency UPD glucosyltransferase mutant is introduced; biosynthetic pathways of gastrodin and p-hydroxybenzyl alcohol are organically combined together; yield of gastrodin is obviously improved; the highest yield of gastrodin can reach 265mg / L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Escherichia coli which utilizes glucose to produce p-hydroxybenzyl alcohol or gastrodin and its use. Background technique [0002] Gastrodia elata is the stem of the orchid plant Gastrodia elata B1., which is a commonly used and precious traditional Chinese medicine. The plant Gastrodia elata grows in sparse forests, in open spaces, forest edges, and shrub edges, at an altitude of 400-3200 meters. It has been rated as a vulnerable species by the International Union for Conservation of Nature (IUCN), and has been included in the "Endangered Species of Wild Animals and Plants". In Appendix II of the Convention on International Trade (CITES), it is also included in China's "List of National Key Protected Wild Plants (Second Batch)", and is a Class II protected plant. Its rhizome is used medicinally to treat dizziness, numbness of limbs, convulsions ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/22C12P19/44C12R1/19
Inventor 刘涛白艳芬殷华毕慧萍庄以彬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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