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Gene engineering bacterium for catalyzing citral to produce citronellal and construction method thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of genetically engineered bacteria that biocatalyze citral to generate citronellal and its construction

Inactive Publication Date: 2008-05-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are documents abroad about the use of microorganisms to catalyze the hydrogenation of citral to generate citronellal, there are no relevant literature and patent reports in China, and the microorganisms used in the existing microorganisms to catalyze the hydrogenation of citral to generate citronellal are all wild Bacteria, there is no report of using genetically engineered bacteria to catalyze the hydrogenation of citral to generate citronellal, therefore, it is necessary to study this

Method used

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  • Gene engineering bacterium for catalyzing citral to produce citronellal and construction method thereof
  • Gene engineering bacterium for catalyzing citral to produce citronellal and construction method thereof

Examples

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Embodiment 1

[0033] Example 1 , Construction of genetically engineered bacteria

[0034] 1.1. Primer design

[0035] According to the sequence of the NADH oxidase gene gye in NCBI Genbank, the PCR primers Pgye-1 and Pgye-2 sequences for amplifying the gye gene were designed as follows:

[0036] Pgye-1: 5'-CGAC ATATG CCAACCCTGTTCGATC-3';

[0037] Pgye-2: 5'-TTAG AATTC TCAGTTGGGGCCGGAGGTG-3'.

[0038] In order to facilitate enzyme digestion and connection with the expression vector, an Nde I restriction site was introduced into the primer Pgye-1, and an EcoR I restriction site was introduced into the primer Pgye-2. The restriction sites are respectively underlined in the sequences.

[0039] 1.2. Cloning of gene gye

[0040] The total DNA of Gluconobacter oxidans (DSM2003) was obtained with a bacterial DNA extraction kit, and the gdh gene was amplified by PCR using it as a template and Pgye-1 and Pgye-2 as primers. The specific conditions were as follows:

[0041] Reaction system: 1 ...

Embodiment 2

[0050] Example 2 , Genetic engineering bacteria fermentation expression and product detection

[0051] 2.1. Fermentative expression and purification of genetically engineered bacteria BL21 / gye

[0052] The genetically engineered bacteria BL21 / gye obtained in Example 1 was inoculated into LB medium, cultivated to the logarithmic growth phase at 37° C., added IPTG to a final concentration of 1 mmol / L, and continued to cultivate for 6 hours.

[0053] Bacteria were collected by centrifugation, and the bacteria were broken by ultrasonication. The supernatant was collected by centrifugation and purified with a His-Tag affinity column. The obtained purified product was subjected to SDS-PAGE electrophoresis. It is expected that the band size of NADH oxidase is consistent, which proves that the constructed genetically engineered strain BL21 / gye can ferment and express NADH oxidase.

[0054] 2.2. Detection of fermentation products

[0055]Add the purified NADH oxidase protein obtain...

Embodiment 3

[0056] Example 3 , Production of citronellal by genetically engineered bacteria BL21 / gye

[0057] The strain used in this example is the genetically engineered bacterium BL21 / gye obtained in Example 1.

[0058] The medium formula used in this example is: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, kanamycin 0.05g / L, pH 7.0.

[0059] Insert the genetically engineered bacteria BL21 / gye into a 500mL shake flask filled with 200mL medium, culture at 37°C and 250rpm, and take samples regularly to detect OD 650 value, when OD 650 When it is 0.4, add IPTG to a final concentration of 1 mmol / L, and induce culture for 14 hours.

[0060] Add citral to the shake flask to a final concentration of 10% by weight and volume, add acetonitrile to a final concentration of 10% by weight to volume, and react at 35° C. and 250 rpm for 4 hours.

[0061] Get reaction solution, obtain citronellal with chloroform extraction separation and purification, measure and adopt gas chromatogr...

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Abstract

The invention provides genetic engineering bacteria for catalyzing citral to generate citronellal and a construction method thereof. Recombinant expression plasmid provided by the invention is to clone gene gye with NADH oxidase on a suitable vector. The genetic engineering bacteria provided by the invention is transformed into the recombinant expression plasmid in escherichia coli. The invention provides new genetic engineering bacteria for producing citronellal, catalyzes hydrogenation of citral selectively to generate citronellal by employing the expression product NADH oxidase of the gene gye of NADH oxidase, which simplifies manufacturing technique and improves production efficiency. The invention sets a precedent of utilizing genetic engineering bacteria to produce citronellal, as a result, the normal escherichia coli is provided with the function of catalyzing citral to generate citronellal, which provides a new thinking for constructing the genetic engineering bacteria for produce citronellal and lays a foundation for achieving a more optimal engineering bacteria.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a genetically engineered bacterium and a construction method thereof, more specifically to a genetically engineered bacterium for biocatalyzing citral to generate citronellal and a construction method thereof. Background technique [0002] Citronellal is an important isolated fragrance, which is widely used in the flavoring of beverages, candies, food, etc. and the preparation of other essences, and is also a raw material for the synthesis of other fragrances. Industrially, it is mainly separated from essential oils such as citronella oil and eucalyptus oil. It can also be obtained by catalytic hydrogenation of citral or by hydrogenation of β-citronellol. [0003] The reaction formula of microbial catalyzed hydrogenation of citral to generate citronellal is as follows: [0004] [0005] Its mechanism is to selectively catalyze hydrogenation by oxidoreductase prod...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/53C12N15/66C12N1/21C12N9/04C12P7/24
Inventor 魏东芝杨雪鹏殷波魏国栋马昱澍
Owner EAST CHINA UNIV OF SCI & TECH
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