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Efficient biosynthesis method for transforming low-value compounds into caffeic acid

A technology of biosynthesis and caffeic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of no significant increase in yield, unsatisfactory effect, and low substrate conversion rate of caffeic acid production, reaching Effects of cheap mass production and easy mass production

Active Publication Date: 2017-03-15
JIANGNAN UNIV
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  • Description
  • Claims
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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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  • Efficient biosynthesis method for transforming low-value compounds into caffeic acid
  • Efficient biosynthesis method for transforming low-value compounds into caffeic acid
  • Efficient biosynthesis method for transforming low-value compounds into caffeic acid

Examples

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Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction method of recombinant Escherichia coli

[0032] The genes EhTPL and TcXAL were optimized and synthesized by GenScript Biotechnology Co., Ltd., and cloned into pUC57-Simple. The recombinant plasmids were named pUC57-EhTPL and pUC57-TcXAL, respectively. pET28a(PB) is an ePathBrick expression vector constructed on the basis of pET-28a(+). The vector contains homologous enzymes Avr II, Xba I, Spe I and Nhe I, namely the downstream Sal I. Different ePathBrick strategies can be used to construct Co-expression constructs. The structure of pET28a(PB) is as follows figure 2, the DNA sequence is SEQ ID NO.5. The recombinant vectors pUC57-EhTPL, pUC57-TcXAL and the expression vector pET28a(PB) were digested with restriction endonuclease Bam HI / Hind III respectively, the uncut products were separated by agarose gel electrophoresis, and the target gene EhTPL (1371bp) was recovered respectively , TcXAL (2070bp) and expression vector pET28a(PB) (5371bp). Ac...

Embodiment 2

[0037] Example 2 Analysis of recombinant Escherichia coli strCL-1 catalyzing the synthesis ability of levodopa

[0038] In order to verify the ability of recombinant escherichia coli E.coli strCL-1 in Table 1 to catalyze the synthesis of levodopa from catechol, the transformation method described in Example 1 was used to transform the escherichia coli BL21 ( DE3) As a blank control, PBS (50mM, pH 7.0) was used as the reaction medium, reacted at 37°C and 220rpm, samples were taken at specific time points, and the synthesis of levodopa was determined.

[0039] The results showed that levodopa was synthesized in the reaction system catalyzed by E.coli strCL-1, but levodopa was not detected in the blank control. This result verified the ability of EhTPL to catalyze the synthesis of levodopa from catechol, and the synthesis process could not proceed spontaneously in the absence of tyrosine benzene lyase. The chromatogram and mass spectrum of levodopa are as follows Figure 4 , wh...

Embodiment 3

[0040] Example 3 Recombinant Escherichia coli catalyzes the ability to synthesize caffeic acid from catechol

[0041] In order to verify the ability of the recombinant Escherichia coli listed in Table 1 to transform catechol, sodium pyruvate and ammonium chloride into caffeic acid, the collected bacteria were washed with 25 mL of PBS, centrifuged and resuspended in an equal volume of PBS (50 mM, pH 7.0), the cell concentration is OD 600 =18±1. Simultaneously add 0.65M NH 4 Cl, 50 mM sodium pyruvate and 50 mM catechol were used as substrates for the reaction, and the reaction was carried out on a constant temperature shaker at 37° C. and 220 rpm. Escherichia coli BL21(DE3) containing empty plasmid pET28a(PB) was used as blank control. Samples were taken at specific time points to determine the synthesis of caffeic acid. Take the maximum production of caffeic acid in 0-10 hours as the corresponding maximum production of the strain.

[0042] The results showed that the recom...

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Abstract

The invention discloses an efficient biosynthesis method for transforming low-value compounds into caffeic acid, and belongs to the field of biochemical engineering. The method includes that tyrosine phenol lyase takes catechol, pyruvic acid and ammonia as substrates to synthesize levodopa, and tyrosine amino lyase converts levodopa into trans-caffeic acid and NH3. Since catechol, sodium pyruvate and ammonium chloride are all relatively cheap compounds, the efficient biosynthesis method is created for transforming low-value compounds into caffeic acid and is easy for large-scale production. Compared with a chemical synthesis method, the efficient biosynthesis method has the advantages that the product of the method is single trans-caffeic acid, and further separation of structural isomers is not required.

Description

technical field [0001] The invention relates to a high-efficiency biosynthesis method for converting low-value compounds into caffeic acid, which 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 of phenolic hydroxyl 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 an oxidation mechanism; caffeic acid has immunomodulatory and anti-inflammatory activities; caffeic acid can also act as an antioxidant, which is superior to other natural compounds; in addition, caffeic acid also has antiviral, antidepressant , treatment of diabetes and other activities. [0003] As a key intermediate metabolite of lignin synthesis, caffeic acid exists in almost all plants. The pathway...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N1/21C12R1/19
CPCC12N9/88C12P7/42C12Y401/03
Inventor 周景文陈坚吕永坤堵国成
Owner JIANGNAN UNIV
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