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Engineering bacterium used for co-production of geraniol and nerol, and construction method and application of engineering bacterium

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

Active Publication Date: 2021-02-23
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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  • Engineering bacterium used for co-production of geraniol and nerol, and construction method and application of engineering bacterium
  • Engineering bacterium used for co-production of geraniol and nerol, and construction method and application of engineering bacterium
  • Engineering bacterium used for co-production of geraniol and nerol, and construction method and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 is used for the construction of the engineering bacteria of co-production geraniol and nerol:

[0056] (1) Clone 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) Recombinant bacteria transformation: Transform E.coli BL21(DE3) competent cells with the pYJM14 plasmid and the recombinant plasmid pT2a obtained in step (2) 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] Extract RNA from tobacco leaf tissue, use reverse transcriptase to convert RNA into cDNA template, use cDNA obtained by reverse transcription reaction as template, forward primer: 5'-ATGGCCACCTCCATAAGACCTGCAA-3', reverse primer: 5'-TTATAGGGATGGATTG...

Embodiment 2

[0070] Example 2 Application of engineering bacteria for co-production of geraniol and nerol in fermentation co-production of geraniol and nerol:

[0071] Inoculate the single colony of engineering bacteria prepared on the solid LB plate obtained in Example 1 in the LB seed liquid medium, and add a final concentration of 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, grow at 37 ° C for 8-12 h, Obtain the primary seed liquid; transfer the obtained primary seed liquid to 500mL fermentation shake flasks according to 2% (wt) inoculum respectively, containing 100mL M9 seed medium, to obtain the secondary seed liquid. Inoculate the secondary seed liquid into a 5L small-scale fermenter containing 2L fermentation medium at an inoculum amount of 5%wt. The ventilation rate is 1-2vvm, the dissolved oxygen is controlled at about 0-20%, the rotation speed is 400-1000rpm, and cultured at 37°C When the OD600 is about 12, add IPTG with a final concentration of 0.3mM, induce expression at ...

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Abstract

The invention discloses an engineering bacterium used for co-production of geraniol and nerol, and a construction method and application of the engineering bacterium, and belongs to the technical field of bioengineering. The construction method of the engineering bacterium includes the following steps: introducing a tobacco geraniol and nerol synthetase gene into a receptor strain capable of generating geranyl pyrophosphate to obtain a recombinant bacterium; and obtaining the engineering bacterium used for producing the geraniol and the nerol from the recombinant bacterium. According to the engineering bacterium, a bioengineering technology is successfully applied to realize the heterologous reconstruction of a geraniol and nerol biosynthetic pathway and the co-production of products, andthe yield of the geraniol and the yield of the nerol reach 137.3 mg / L and 62.8 mg / L respectively by a shake-flask fermentation method; and meanwhile, the yield of geraniol and the yield of nerol are increased by about 10 times through a high-density fermentation technology of recombinant strains.

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, as well as a preparation method and application thereof. Background technique [0002] Geraniol (also known as geraniol, whose 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 a non-cyclic monoterpene alcohol compound, which is one of the main components of essential oils such as rose oil, martin balm and citronella oil. Geraniol is the main component of rose oil (accounting for about 40% to 60%). It has the fragrance of roses and is a precious spice. Neroli has a pleasant aroma of roses and orange blossoms. The aroma is relatively mild, with a slight lemon-like fruity aroma. Compared with geraniol, its aroma is softer, more beautiful, and relatively clear. Geraniol and nerol can be used in flavors and food flavors. T...

Claims

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Application Information

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Patent Type & Authority Applications(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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