Method for efficient synthesis of caffeic acid with catechol as substrate

A technology of catechol and caffeic acid, which is applied in the field of high-efficiency synthesis of caffeic acid, can solve the problems of not significantly increasing the yield, poor solubility, long production cycle and the like, and achieve the effect of increasing the yield of caffeic acid

Active Publication Date: 2017-04-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the poor solubility of substrates such as tyrosine, the production of caffeic acid and the conversion rate of substrates of this type of engineering bacteria are low.
Using L-tyrosine high-yielding strains as the chassis of caffeic acid heterologous synthesis pathway helps to solve this problem, but the effect is not ideal, which is characterized by a longer production cycle and no significant increase in yield

Method used

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  • Method for efficient synthesis of caffeic acid with catechol as substrate
  • Method for efficient synthesis of caffeic acid with catechol as substrate
  • Method for efficient synthesis of caffeic acid with catechol as substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 The construction method of recombinant Escherichia coli

[0036] (I) Construction of EhTPL and RgTAL co-expression vector

[0037] The genes EhTPL and RgTAL were optimized and synthesized by GenScript Biotechnology Co., Ltd. and cloned into pUC57-Simple. The recombinant plasmids were named pUC57-EhTPL and pUC57-RgTAL respectively. pET28a(PB) is an ePathBrick expression vector constructed on the basis of pET-28a(+), the vector contains homozygous enzymes Avr II, Xba I, Spe I and Nhe I, that is, the downstream Sal I, which can be constructed by ePathBrick strategy. co-expression structure. The structure of pET28a(PB) is as follows figure 2 , the DNA sequence is SEQ ID NO.5. The recombinant vectors pUC57-EhTPL, pUC57-RgTAL and the expression vector pET28a(PB) were digested with restriction enzymes Bam HI / Hind III, respectively, and the uncut products were separated by agarose gel electrophoresis, and the target gene EhTPL (1371bp) was recovered separately....

Embodiment 2

[0047] Example 2 Analysis of the ability of recombinant Escherichia coli strEhTPL to catalyze L-dopa synthesis

[0048] In order to recombine the ability of E. coli strEhTPL to catalyze the synthesis of L-dopa from catechol, the E. coli strCtr containing the empty plasmid pET28a (PB) was used as a blank control, and PBS (50mM, pH 7.0) was used as the reaction medium according to the above transformation method, The reaction was carried out at 37° C. and 220 rpm, and samples were taken at specific time points to determine the synthesis of levodopa.

[0049] The results showed that L-dopa was synthesized in the reaction system catalyzed by strEhTPL, but no L-dopa was detected in the blank control. This result validates the ability of EhTPL to catalyze the synthesis of L-dopa from catechol, and the synthesis cannot proceed spontaneously in the absence of tyrosine benzene lyase. The chromatogram and mass spectrum of levodopa are shown in Figure 4 , where A is the chromatogram o...

Embodiment 3

[0050] Example 3 Analysis of the ability of recombinant Escherichia coli strRgTAL to catalyze caffeic acid synthesis and optimization of substrate concentration

[0051] The recombinant E. coli strRgTAL was cultured, and the expression of RgTAL was induced with 0.5 mM IPTG. The collected cells were washed with 25mL PBS, and resuspended in an equal volume of PBS (50mM, pH 7.0) after centrifugation. The cell concentration was OD. 600 =18±1. At the same time, levodopa with a final concentration of 1 g / L was added as a substrate to carry out the reaction, and the reaction was carried out at 37° C. and a constant temperature shaker at 220 rpm. E. coli strCtr containing the empty plasmid pET-28a(PB) was used as a blank control. Samples were taken at specific time points to determine the synthesis of caffeic acid.

[0052] The results showed that caffeic acid was synthesized in the reaction system catalyzed by strRgTAL, while no caffeic acid was detected in the blank control. Thi...

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Abstract

Belonging to the field of biochemical engineering, the invention discloses a method for efficient synthesis of caffeic acid with catechol as the substrate. A recombinant strain constructed by the invention employs catechol, sodium pyruvate and ammonium chloride to synthesize caffeic acid, the caffeic acid yield can reach 1.51g / L, and catechol, sodium pyruvate and ammonium chloride are all relatively cheap compounds, therefore the method provided by the invention is a caffeic acid biosynthesis method with great potential. Compared with the previous bioconversion method using L-tyrosine as the substrate, the method provided by the invention significantly improves the caffeic acid yield. Compared with the chemical synthesis method, the product of the method is a single trans-caffeic acid, and further separation of an isomer is not needed.

Description

technical field [0001] The invention relates to a method for efficiently synthesizing caffeic acid by using catechol as a substrate, and belongs to the field of biochemical industry. Background technique [0002] Caffeic acid is a high-value aromatic compound, which can be divided into hydroxycinnamic acid in structure, which has two functional groups: phenolic hydroxyl group and acrylic acid. In vivo and in vitro studies have shown that caffeic acid has a range of physiological functions. For example, caffeic acid can inhibit the proliferation of cancer cells through oxidative mechanisms; caffeic acid has immunomodulatory and anti-inflammatory activities; caffeic acid can also act as an antioxidant and is superior to other natural compounds; in addition, caffeic acid also has antiviral, antidepressant properties , the treatment of diabetes and other activities. [0003] As a key intermediate metabolite in lignin synthesis, caffeic acid is present in almost all plants. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N1/21C12R1/19
CPCC12N9/88C12P7/42C12Y401/99002C12Y403/01023
Inventor 周景文陈坚吕永坤堵国成
Owner JIANGNAN UNIV
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