Method for producing hydroxytyrosol

A technology for producing hydroxytyrosol and hydroxytyrosol, applied in the field of bioengineering, can solve the problems of low expression of exogenous protein HpaBC, difficulty in improving the efficiency of hydroxytyrosol, toxicity of hydroxytyrosol bacteria, etc., and achieves good industrial application prospects. , low price, easy availability of raw materials

Pending Publication Date: 2019-02-01
JIANGNAN UNIV
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Problems solved by technology

[0006] Chinese invention patent CN201510242626.8 discloses a monooxygenase gene cluster HpaBC derived from Escherichia coli overexpressed in Escherichia coli to synthesize hydroxytyrosol from scratch using glucose as a substrate; the disadvantage of this scheme is that hydroxytyrosol It is toxic to bacteria, and the expression level of foreign protein HpaBC is low; therefore, it is difficult to improve the efficiency of producing hydroxytyrosol
The disadvantage of this scheme is that a lot of expensive coenzyme pyridoxal phosphate (PLP) and reduced coenzyme I (NADH) need to be added, and the efficiency of adding a hydroxyl group to the benzene ring is significantly reduced, resulting in a very low yield of hydroxytyrosol

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  • Method for producing hydroxytyrosol
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  • Method for producing hydroxytyrosol

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Experimental program
Comparison scheme
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Embodiment 1

[0071] gene cloning

[0072] (1) Primer design

[0073] Design primers for PCR amplification.

[0074] (2) PCR amplification

[0075] According to the instruction manual of the Genomic DNA Purification Kit kit from Takara Company, the genomic DNA of wild strains in the logarithmic growth phase was extracted, and the primers in Table 1 were used to perform PCR amplification using the genome extracted from each corresponding strain as a template. The amplification system is: Prime STARHS DNA Polymerase (2.55U / μL) 0.5 μL, 10×PrimeSTAR Buffer 10 μL, dNTP Mixture (2.5mM each) 4 μL, template DNA 1 μL, Up primer (20 μM) 1 μL, Down primer (20 μM) 1 μL ,ddH 2 O to make up to 50 μL. The amplification program is: 94°C, 10min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min, a total of 30 cycles; 72°C, 10min. PCR products were sent to BGI for sequencing.

[0076] Later, L-α-amino acid transaminase genes lparo and lcaro were respectively cloned from Lactobacillus plantarum ATCC 14917 and Lactob...

Embodiment 2

[0084] For the screening of L-α-amino acid transaminase, a variety of recombinant engineering bacteria containing L-α-amino acid transaminase genes were obtained from Example 1, and expressed in Escherichia coli BL21 (DE3). Induced expression method: the recombinant Escherichia coli is transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in the amount of 2% by volume ratio, when the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.4mM, induce expression and culture at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes. Collect the cells, break the cells to measure the activity of the crude enzyme solution, and compare the activities of various enzymes when α-ketoglutarate is used as the receptor. (11): 1346-1358.) The method measures the activity of L-α-amino acid transaminase, and the results are shown in Table 2. Therefore, it is best to choos...

Embodiment 3

[0088] The screening of L-glutamic acid dehydrogenase obtains the recombinant engineered bacterium of multiple gene containing L-glutamic acid dehydrogenase from embodiment 1, according to the induced expression method in embodiment 2, and in E.coli Expressed in BL21(DE3). According to the literature (cloning, expression and enzyme activity determination of Bacillus natto glutamic acid dehydrogenase gene. Shanghai Jiaotong University Journal of Agricultural Sciences, 2010, 1:82-86.), the activity of the crude enzyme solution was determined by breaking the cells. The method described above was used to measure the activity of L-glutamate dehydrogenase with NAD as a coenzyme, and the results are shown in Table 3. Therefore, it is best to choose L-glutamate dehydrogenase bsgdh derived from Bacillus subtilis for the production of hydroxytyrosol.

[0089] Table 3 Activity comparison of different L-glutamate dehydrogenases

[0090] Recombinant bacteria

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Abstract

The invention discloses genetic engineering bacteria and belongs to the technical field of bioengineering. With the introduction of L-alpha-amino acid transaminase, L-glutamic acid gene, alpha-keto acid decarboxylase gene and alcohol dehydrogenase, the genetic engineering bacteria can be applied to biosynthesis of hydroxytyrosol. The invention also discloses a construction method and application of the recombinant Escherichia coli genetic engineering bacteria. And further, substrate transport is promoted and decomposition of the product is reduced by knocking out or enhancing the expression ofrelated genes on the Escherichia coli genome, and the production efficiency of recombinant bacteria is improved. The method for production of hydroxytyrosol by conversion of the recombinant bacteriahas characteristics of simple operation, low cost and high synthesis efficiency of the product, and has a good industrial prospect.

Description

technical field [0001] The invention relates to a method for producing hydroxytyrosol, which belongs to the technical field of bioengineering. Background technique [0002] Hydroxytyrosol (3,4-Dihydroxyphenylethanol, Hydroxytyrosol, 3,4-Dihydroxyphenylethanol, 2-(3,4-Dihydroxyphenyl)ethanol), is a natural polyphenol compound with strong antioxidant activity , mainly in the form of esters in the fruits and branches of olives. The phenyl ring of hydroxytyrosol is connected with two hydroxyl groups, which are its main antioxidant active groups, and because of its inherent antioxidant activity, it can prevent the occurrence of various diseases. In addition, hydroxytyrosol can also eliminate free radicals in the body, restore the health of the internal organs of the human body, prevent brain failure, and delay aging. [0003] Studies in recent years have shown that hydroxytyrosol has obvious effects on a variety of diseases. Hydroxytyrosol can regulate glucose and lipid metabol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N1/21C12R1/19
CPCC12N9/0006C12N9/0016C12N9/1096C12N9/88C12P7/22
Inventor 蔡宇杰刘金彬李朝智丁彦蕊白亚军郑晓晖
Owner JIANGNAN UNIV
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