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Escherichia coli expression strain for high yield of geraniol glucoside and application thereof

A technology of Escherichia coli and glucoside, applied in the field of genetic engineering, can solve the problems of complex synthesis steps, low yield, environmental pollution, etc., and achieve the effects of reliable quality, efficient synthesis and good application prospects

Active Publication Date: 2019-10-11
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In the prior art, the production of geraniol glucoside mostly adopts the method of chemical synthesis using geraniol as a substrate, and the chemical synthesis often has problems such as environmental pollution, complex synthesis steps, and low yield.
However, there is no report on the de novo synthesis of geraniol glucoside engineering strains from simple carbon sources such as glucose

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  • Escherichia coli expression strain for high yield of geraniol glucoside and application thereof
  • Escherichia coli expression strain for high yield of geraniol glucoside and application thereof
  • Escherichia coli expression strain for high yield of geraniol glucoside and application thereof

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preparation example Construction

[0030] The present invention also provides a preparation method of the Escherichia coli expression strain with high-yield geraniol glucoside, which comprises the following steps: preparing co-expressing acetoacetyl-CoA thiolase gene atoB and hydroxymethylglutaryl-CoA synthase gene HMGS, hydroxymethylglutaryl coenzyme A reductase gene tHMGR, mevalonate kinase gene ERG12, phosphomevalonate kinase gene ERG8, pyrophosphate mevalonate decarboxylase gene MVD1, isopentenyl pyrophosphate The first expression vector of phosphate isomerase gene idi and geranyl pyrophosphate GPP synthase gene ispA*; preparation of co-expression of geraniol synthase gene GES, geranyl pyrophosphate GPP synthase gene ERG20* and glycosyltransferase A second expression vector for the gene AdGT4; the first expression vector and the second expression vector are jointly transformed into Escherichia coli to obtain an Escherichia coli expression strain with high yield of geraniol glucoside.

[0031] The present inven...

Embodiment 1

[0058] Example 1 Construction of Escherichia coli expression strain with high yield of geraniol glucoside

[0059] (1) Construction of E. coli expression vector P1 (pBbA5c-GPP)

[0060] ispA* site-directed mutagenesis: pBbA5c-MM is digested with restriction enzymes Xhol and HindIII, the 2.1kbispA fragment and 11kb plasmid fragment with ispA cut out are recovered from the gel; the ispA fragment is ligated with the same digested pUC19 with T4DNA ligase The ligation product was chemically transformed into Escherichia coli DH5α competent, and the transformant was picked to 3ml LB plus chloramphenicol-resistant liquid medium, cultured at 37°C for 8-12 hours, centrifuged at 7000rpm for 1min, the bacteria were collected, and the plasmid was extracted. The plasmid was digested with restriction enzymes Xhol and HindIII to verify that the plasmid was 4.8kb pUC19-ispA. Then use the mutant primers ispA*-P1, ispA*-P2, and use the plasmid pUC19-ispA as the template for PCR amplification. The PC...

Embodiment 2

[0070] Example 2 Liquid fermentation of Escherichia coli expression strain B-GesA of geraniol glucoside and control strain B-Ges

[0071] When the E. coli expression strain B-GesA is fermented and cultivated, the method of the fermentation culture is to add the E. coli expression strain B-GesA in an LB liquid medium with 20 mg / L chloramphenicol and 100 mg / L ampicillin 37 Incubate at ℃ for 16 hours, then transfer the culture solution to the LB liquid medium with the same antibiotics at a transfer volume of 2% by volume. 600 When it is 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1mM, induce culture at 16°C for 24 hours, centrifuge, and resuspend in an equal volume of M9 liquid medium. Incubate at 30°C for 48 hours.

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Abstract

The invention provides an Escherichia coli expression strain for high yield of geraniol glucoside, and belongs to the technical field of genetic engineering. The Escherichia coli expression strain jointly expresses an acetoacetyl-CoA thiolase gene, a methyl glutaryl coenzyme A synthetase gene, a HMG-CoA reductase gene, a mevalonate kinase gene, phosphomevalonate kinase gene, a pyrophosphate mevalonate decarboxylase gene, an isopentenyl diphosphate isomerase gene, a geranyl pyrophosphate GPP synthetase ispA*, a geraniol synthetase gene, a geranyl pyrophosphate GPP synthetase ERG20* and a glycosyltransferase gene. The yield of geraniol glucoside produced from the Escherichia coli expression strain is high, reliable in mass and good in application prospect.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an Escherichia coli expression strain with high yield of geraniol glucoside and its application. Background technique [0002] Geraniol is an acyclic monoterpene compound with a mild and elegant rose aroma. It is naturally present in more than 200 essential oils such as rose essential oil, geraniol oil, citronella oil and palmarosa oil. Geraniol is widely used in daily floral flavors, edible flavors, ester flavors, etc. It is also a raw material for the manufacture of vanillin, vanillin, citral, hydroxyvanillin, ionone and vitamin A. Geraniol is used as a medicine to relieve asthma, improve lung ventilation and reduce airway resistance. It is clinically used to treat chronic bronchitis. Geraniol has the advantages of improving the immune function of the body, and has the advantages of fast onset and small side effects. It has been used as a chemopreventive agen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/44C12R1/19
CPCC12N9/0006C12N9/1025C12N9/1029C12N9/1048C12N9/1205C12N9/1229C12N9/88C12N9/90C12P19/44C12Y101/01088C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002
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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