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A kind of engineering bacteria and method for producing Danshensu

A technology of danshensu and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of high cost of 3,4-dihydroxyphenylpyruvate intermediate, increase the complexity of catalytic reaction, increase the cost of industrial application, etc., and achieve good industrial application Foreground, simple process, and easy-to-obtain raw materials

Active Publication Date: 2021-03-26
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

The cost of these two methods for preparing 3,4-dihydroxyphenylpyruvate intermediates is relatively high, and the operation is complicated, requiring the use of glucose or other hydrogen donors, which increases the cost of industrial applications and increases the complexity of the catalytic reaction

Method used

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  • A kind of engineering bacteria and method for producing Danshensu
  • A kind of engineering bacteria and method for producing Danshensu
  • A kind of engineering bacteria and method for producing Danshensu

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Experimental program
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Effect test

Embodiment 1

[0052] 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. The specific enzymatic activity of the three mutants of L-phenylalanine dehydrogenase to levodopa dehydrogenation was measured (that is, with levodopa as substrate and NAD as coenzyme, dehydrogenation generates 3,4-dihydroxyphenylpyruvate ) were 20.2, 18.9, 6.4 U / mg, respectively. Taking L-phenylalanine dehydrogenase with the accession NO of the amino acid sequence on NCBI as BAA08816.1 as a control,...

Embodiment 2

[0054] Recombinant Escherichia coli construction: first, the genes encoding α-hydroxycarboxylic acid dehydrogenase and L-phenylalanine dehydrogenase were connected to the plasmid. The double-gene co-expression recombinant plasmid was obtained, the plasmid was transformed into E. coli Escherichia coli BL21 (DE3), and the positive transformant was obtained by screening with an antibiotic plate, that is, the recombinant E. coli was obtained.

[0055]After the induced expression of recombinant Escherichia coli was completed, the bacterial cells were collected, and the final cell concentration was 80g / L in a 100ml reaction system containing 1g / L NAD, 20g / L levodopa, pH 8.0, and reacted at 35°C. The shaker rotates at 50 rpm for 12 hours. After the conversion, the yield and configuration of Danshensu were determined by liquid chromatography.

[0056] The recombinant bacteria in Table 1 were used in the above transformation system, and the results are shown in Table 1.

[0057] The ...

Embodiment 3

[0061] According to the inducible expression method described in Example 1, the cells were collected after the induced expression of Escherichia coli BL21(DE3) / pETDuet-1-csldhd-bspde2 was completed, and in a 100ml reaction system, the final cell concentration was 100g / L, and levodopa 1g / L, pH 9.0, temperature 40°C, shaker speed 50 rpm; transformation time 1 hour. As a result of the measurement, the concentration of S-danshensu was 821mg / L, e.e%>99.9.

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Abstract

The invention discloses an engineering bacterium and a method for efficiently producing Danshensu, and belongs to the technical field of bioengineering. The invention constructs a novel double-enzymeco-expression genetically engineered bacterium which can be applied to the production of optically pure Danshensu. The (D / L)-[alpha]-hydroxy carboxylic acid dehydrogenase selected by the invention hasthe characteristics of poor substrate specificity and high optical specificity, and optically pure R-Danshensu and S-Danshensu can be produced. By use of the method for producing Danshensu through the transformation of recombinant bacteria, a process is simple, raw materials can be easily obtained, impurities are few, and the method has an important industrial application value.

Description

technical field [0001] The invention relates to an engineering bacterium and a method for producing danshensu, belonging to the technical field of bioengineering. Background technique [0002] Danshensu extracted from Danshen, scientific name R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid, D-(+)-β-(3,4-dihydroxyphenyl ) lactic acid, English name: Danshensu, D-DSS, R-DSS, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lactic acid, (R)-(+)-3-(3 ,4-Dihydroxyphenyl)-2-hydroxypropanoic acid is a dextrophenolic acid compound. Natural levodanshensu does not currently exist. [0003] Danshensu is an important active ingredient in the water extract of Danshen, and its structure was obtained and identified in China in 1980 from the water extract of Danshen (Study on the water-soluble active ingredients of Danshen, Ⅱ.D(+)β(3,4-di The structure of hydroxyphenyl) lactic acid, Shanghai First Medical College Journal, 1980, 05 (7), 384-385), various studies have shown that Danshensu has importan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N1/21C12P7/42C12R1/25C12R1/225C12R1/145
CPCC12N9/0018C12P7/42C12Y104/0102
Inventor 蔡宇杰熊天真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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