Strain and method for producing hydroxytyrosol

A technology of hydroxytyrosol and alcohol dehydrogenase, which is applied in the field of microbial genetic engineering, can solve problems such as low cost, differences between batches, and obstacles to industrialization, and achieve the goals of reducing by-products, avoiding losses, and reducing costs Effect

Pending Publication Date: 2022-06-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Plant extraction uses oleuropein as raw material, which is extracted from the waste of olive oil production. However, due to the complexity and low content of the extracted raw materials, the cost is high, the purity of the obtained hydroxytyrosol is low, and there are differences between batches. Issues of variance and seasonality
[0005] Since 1949, For the first time, 3,4-dihydroxyphenylacetic acid was used to chemically synthesize hydroxytyrosol, but the catalyst lithium aluminum hydride used was poor in stability, and the cost of the substrate was high, which led to obstacles in industrialization ( β-(3,4-Dioxyphenyl)- Justus Liebigs Annalen der Chemie.1949;563(1):86-93.)
In 2018, Ziosi and others used catechol and glyoxal dimethyl acetal to synthesize hydroxytyrosol. Although catechol has a wide range of sources, the cost of glyoxal dimethyl acetal is not low. In the process of two-step reduction, heavy metal palladium was used to catalyze (ATtwo-Step Process for the S...

Method used

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  • Strain and method for producing hydroxytyrosol
  • Strain and method for producing hydroxytyrosol
  • Strain and method for producing hydroxytyrosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Cloning of Example 1 Gene

[0062] (1) Primer design

[0063] Design primers for PCR amplification.

[0064] Table 1 Primer sequence list

[0065]

[0066]

[0067] Note: The underline is the restriction endonuclease site

[0068] (2) PCR amplification

[0069] According to the instruction manual of the Genomic DNA Purification Kit of Takara Company, the genomic DNA of the wild strain in logarithmic growth phase was extracted, and the genome extracted from each corresponding strain was used as a template for PCR amplification. Amplification system: Prime STARHS DNA Polymerase (2.55U / μL) 0.5μL, 10×PrimeSTAR Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Template DNA 1μL, Up primer (20μM) 1μL, Down primer (20μM) 1μL , ddH 2 O make up to 50 μL. The amplification program was: 94°C, 30sec; 55°C, 30sec; 72°C, 2min, a total of 30 cycles: 72°C, 5min. PCR products were sent for sequencing.

[0070] cvta, bata, and pdta were cloned from Chromobacterium violaceum, Brucel...

Embodiment 2

[0077] Example 2 Expression of ω-transaminase from different sources

[0078] The recombinant Escherichia coli expressing different sources of ω-transaminase constructed in Example 1 was cultivated to obtain seed liquid, and the seed liquid was transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L in an amount of 2% by volume respectively). L, NaCl 10g / L), when the cell OD 600 After reaching 0.6-0.8, IPTG with a final concentration of 0.4 mM was added to induce expression and culture at 25°C for 15 h. After induction of expression, cells were collected by centrifugation at 4°C, 8000 rpm, and 20 minutes. After ultrasonication, the supernatant was centrifuged at 4°C, 8000 rpm for 20 minutes to obtain the crude enzyme solution, and the activity of the crude enzyme solution was measured. The results are shown in Table 2. The ω-transaminase derived from Chromobacterium violaceum was more effective for deamination of dopamine.

[0079] Table 2 ω-transaminase act...

Embodiment 3

[0082] Example 3 Expression of alanine dehydrogenases from different sources

[0083] Recombinant Escherichia coli expressing bsaladh (GenBank: QJR47777.1), haaladh (GenBank: RDU72374.1), and mtaladh (GenBank: WP_003899477.1) derived from Bacillus subtilis, Helicobacteraurati, and Mycobacterium tuberculosis were constructed in the same manner as in Example 1. , and according to the same method in Example 2, the recombinant E. coli was cultured to induce the expression of the enzyme. Determination of crude enzyme activity. The results are shown in Tables 3 and 4, and the alanine dehydrogenase derived from Bacillus subtilis was more effective in the conversion reaction of hydroxytyrosol.

[0084] Table 3 Oxidative activity of alanine dehydrogenase

[0085] Recombinant bacteria Activity (U / mL) E.coli BL21 / pET-28a-bsaladh 28.1 E.coli BL21 / pET-28a-haaladh 25.8 E.coli BL21 / pET-28a-mtaladh 14.0

[0086] Table 4 Reductase activity of alanine dehydr...

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Abstract

The invention discloses a strain and a method for producing hydroxytyrosol, and belongs to the technical field of bioengineering. According to the present invention, the recombinant cell for expressing the omega-transaminase, the alanine dehydrogenase and the alcohol dehydrogenase is constructed, and the appropriate RBS strength is adjusted, such that the efficiency of the recombinant escherichia coli cell for catalyzing the dopamine to synthesize the hydroxytyrosol is improved, the yield of the hydroxytyrosol after the reaction for 24 h can achieve 146 g/L, and the good industrial application prospect is provided.

Description

technical field [0001] The invention relates to a strain and a method for producing hydroxytyrosol, belonging to the technical field of microbial genetic engineering. Background technique [0002] Hydroxytyrosol, a natural antioxidant, is considered to be one of the most effective natural antioxidants known, mainly derived from olive oil. Traditional olive oil has been regarded as a very sacred food in Europe because of its various beneficial effects on human replacement. Later, scholars researched that the main components of its efficacy were tyrosol and hydroxytyrosol. And around 2012, hydroxytyrosol was approved by EC Regulation 432 / 2012 as an important part of "healthy olive oil polyphenols", and stipulated that the content of hydroxytyrosol and its derivatives in olive oil should be greater than 5mg / 20g. Due to its many benefits, hydroxytyrosol has been given the name of the supernutrient. [0003] In the prior art, the preparation of hydroxytyrosol is mainly extracte...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/31C12P7/22C12R1/19
CPCC12N9/0016C12N9/1096C12N9/0006C07K14/295C12N15/52C12Y104/01001C12Y206/01C12P7/22
Inventor 蔡宇杰燕毅丁彦蕊白亚军郑晓晖
Owner JIANGNAN UNIV
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