A kind of engineering bacteria for co-producing geraniol and nerol and its construction method and application

A construction method, the technology of nerolidol, applied in the field of bioengineering, can solve problems such as harsh conditions, environmental pollution by chemical synthesis methods, and impact on downstream applications of products, and achieve the effect of cheap raw materials

Active Publication Date: 2022-08-05
TOBACCO RES INST CHIN AGRI SCI ACAD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis method has problems such as environmental pollution and harsh conditions, and the chemical synthesis product is "unnatural". As a food additive, consumers' acceptance is low, which affects the downstream application of the product

Method used

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  • A kind of engineering bacteria for co-producing geraniol and nerol and its construction method and application
  • A kind of engineering bacteria for co-producing geraniol and nerol and its construction method and application
  • A kind of engineering bacteria for co-producing geraniol and nerol and its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Construction of engineering bacteria for co-production of geraniol and nerol:

[0056] (1) Cloning the tobacco geraniol and nerol synthase gene tps2a;

[0057] (2) Construction of the expression vector: the nerol synthase gene tps2a was connected between the BglII and XhoI double restriction sites of the pYJM26 plasmid to obtain the recombinant plasmid pT2a;

[0058] (3) Transformation of recombinant bacteria: The pYJM14 plasmid and the recombinant plasmid pT2a obtained in step (2) were transformed into E.coli BL21 (DE3) competent cells to obtain engineering bacteria for co-production of geraniol and nerol.

[0059] Specifically:

[0060] (1) Cloning of tobacco geraniol and nerol synthase gene tps2a

[0061] RNA from tobacco leaf tissue was extracted, and reverse transcriptase was used to convert RNA into cDNA template. The cDNA obtained by reverse transcription reaction was used as template. The forward primer was: 5'-ATGGCCACCTCCATAAGACCTGCAA-3', and the r...

Embodiment 2

[0070] Embodiment 2 is used in the application of the engineering bacteria that co-produces geraniol and nerol in the fermentation co-production of geraniol and nerol:

[0071] The single colony of engineered bacteria prepared on the solid LB plate obtained in Example 1 was inoculated into the LB seed liquid medium, and the final concentrations of 100 μg / mL ampicillin and 34 μg / mL chloramphenicol were added, and the cells were grown at 37°C for 8-12 h. The first-grade seed liquid was obtained; the obtained first-grade seed liquid was transferred to a 500 mL fermentation shake flask at a 2% (wt) inoculation amount, containing 100 mL of M9 seed medium, to obtain a second-grade seed liquid. The secondary seed liquid was inoculated into a 5L small fermenter containing 2L fermentation medium at an inoculation amount of 5%wt, the ventilation rate was 1-2vvm, the dissolved oxygen was controlled at about 0-20%, the rotation speed was 400-1000rpm, and the culture was carried out at 37°C...

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Abstract

The invention discloses an engineering bacterium for co-producing geraniol and nerol, a construction method and application thereof, and belongs to the technical field of biological engineering. The construction method of the engineering bacteria comprises the following steps: introducing tobacco geraniol and nerol synthase genes into a recipient strain capable of producing geranyl pyrophosphate to obtain recombinant bacteria; obtaining the recombinant bacteria from the recombinant bacteria Engineered bacteria for the production of geraniol and nerol. The present invention successfully utilizes the bioengineering technology to realize the heterologous reconstruction of the biosynthetic pathway of geraniol and nerol and the co-production of the products, and the yields of geraniol and nerol by the shake flask fermentation method reach 137.3 mg / L and 62.8 mg / L respectively. ; At the same time and through the high-density fermentation technology of recombinant strains, the yields of geraniol and nerol were increased by about 10 times.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an engineering bacterium for co-producing geraniol and nerol and a preparation method and application thereof. Background technique [0002] Geraniol (also known as geraniol, scientific name is trans-3,7-methyl-2,6-octadien-1-ol, Geraniol) and its isomer nerol (cis-3, 7-Dimethyl-2,6-octadienol, Nerol) is an acyclic monoterpene alcohol compound, and is one of the main components of essential oils such as rose oil, martin oil and citronella oil. Geraniol is the main component of rose oil (about 40% to 60%), has the fragrance of roses, and is a precious spice. Nerol has a pleasant aroma of rose and orange blossom, and the aroma is relatively mild, with a slightly lemon-like fruity aroma. Its aroma is softer and more beautiful than geraniol, and it is relatively clear. Geraniol and nerol can be used in flavors and edible flavors, are the main agents of rose-based...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/88C12N15/60C12N15/70C12P7/04C12R1/19
CPCC12N9/88C12Y205/0101C12N15/70C12P7/04
Inventor 李依婷张汝兵刘炜咸漠杨爱国
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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