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Engineering bacterium for producing hydroxytyrosol with high efficiency and application of engineering bacterium

A kind of genetically engineered bacteria and gene technology, applied in the field of engineering bacteria for efficient production of hydroxytyrosol, can solve the problems of toxicity of hydroxytyrosol bacteria, increase cost, increase the complexity of catalytic reactions, etc., and achieve good industrial application prospects and low price Inexpensive and readily available raw materials

Active Publication Date: 2019-12-27
卓虹超源生物科技(郑州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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
However, existing methods require the use of glucose or other hydrogen donors, increasing the cost for industrial applications and increasing the complexity of the catalytic reaction

Method used

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  • Engineering bacterium for producing hydroxytyrosol with high efficiency and application of engineering bacterium
  • Engineering bacterium for producing hydroxytyrosol with high efficiency and application of engineering bacterium
  • Engineering bacterium for producing hydroxytyrosol with high efficiency and application of engineering bacterium

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Experimental program
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Effect test

Embodiment 1

[0061] Screening of L-phenylalanine dehydrogenase: According to the random mutation high-throughput screening method of the aforementioned enzymes, error-prone PCR and high-throughput screening platforms were used to obtain three optimal mutants bspde1 and bspde2 for dopa dehydrogenation , bspde3, and sequenced. The amino acid sequences of bspde1, bspde2, and bspde3 are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 respectively, and the nucleotide sequences of the genes encoding these three mutants are shown in SEQ ID NO:1 respectively , SEQ ID NO: 3, shown in SEQ ID NO: 5.

[0062] The specific enzymatic activities of the three mutants of L-phenylalanine dehydrogenase to levodopa dehydrogenation (that is, the conversion of 3,4-dihydroxyphenylpyruvate with levodopa dehydrogenation as a substrate) were 20.2 , 18.9, 6.4 U / mg. Taking L-phenylalanine dehydrogenase with the accession NO of the amino acid sequence on NCBI as BAA08816.1 as a control, the specific activity of t...

Embodiment 2

[0064] Recombinant Escherichia coli construction: first, the genes encoding L-phenylalanine dehydrogenase, α-ketoacid decarboxylase, and alcohol dehydrogenase were connected to the plasmid. The three-gene co-expression recombinant plasmid was obtained, and the plasmid was transformed into E. coli Escherichia coliBL21 (DE3), and the positive transformant was obtained by screening on an antibiotic plate, that is, the recombinant E. coli was obtained.

[0065] After the induction and expression of recombinant Escherichia coli is completed, the bacterial cells are collected. In a 100ml reaction system, the final concentration of cells is 200g / L, the concentration of NAD is 1g / L, the concentration of levodopa is 200g / L, pH 8.5, and react at 35°C. The shaking table rotates at a speed of 50 rpm, and the time is 36 hours. The yield of hydroxytyrosol was determined by liquid chromatography after conversion.

[0066] Table 1 Comparison of various recombinant bacteria

[0067]

Embodiment 3

[0069] During the experiment, it was found that the mutants of bspde had the ability to dehydrogenate other amino acids, so the transformation of some natural amino acids by several engineered bacteria was tested. The results are shown in Table 2, and all amino acids in the table are L-type.

[0070] Construct recombinant E. coli as in Example 2, collect the thalline after the recombinant E. coli is induced and expressed, in a 100ml reaction volume, the cell wet weight is 1g / L, NAD 0.5g / L, substrate concentration is 10g / L (related The substrate is shown in Table 2, the solubility of tyrosine is very small, and it can be reversed while dissolving), pH 8.5, reacted at 35°C, the shaker speed was 50 rpm, and the time was 48 hours. After the conversion, the yield and configuration of Danshensu were determined by liquid chromatography.

[0071] Transformation results of other substrates in table 2

[0072]

[0073]

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Abstract

The invention discloses an engineering bacterium for producing hydroxytyrosol with high efficiency and application of the engineering bacterium, and belongs to the technical field of biological engineering. Genes of L-phenylalaninase dehydrogenase, alpha-keto acid decarboxylase and alcohol dehydrogenase are introduced into the genetic engineering bacterium of the present invention, and the engineering bacterium can be applied to biosynthesis of hydroxytyrosol. The invention also discloses a construction method and application of the genetic engineering bacterium of recombinant Escherichia coli. The method for producing hydroxytyrosol by transforming the recombinant bacterium has the characteristics of simple operation, low cost, high efficiency of product synthesis, and has good industrialization prospects.

Description

technical field [0001] The invention relates to an engineering bacterium for efficiently producing hydroxytyrosol and its application, belonging 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. Hydroxyty...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N9/0018C12N9/88C12N15/52C12P7/22C12Y104/0102C12Y401/01
Inventor 蔡宇杰李朝智刘金彬丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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