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CN-117305195-B - Genetically engineered bacterium for producing cinnamic acid and derivatives thereof, construction method and application thereof

CN117305195BCN 117305195 BCN117305195 BCN 117305195BCN-117305195-B

Abstract

The invention discloses a genetic engineering bacterium for producing cinnamic acid and derivatives thereof, a construction method and application thereof. The genetically engineered bacteria co-express vanillyl alcohol oxidase and oxidoreductase. The genetically engineered bacterium is used as a biocatalyst, and can convert a substrate into cinnamic acid and derivatives thereof. The method for producing cinnamic acid and the derivative thereof has high production efficiency, short reaction period, low cost and good industrialized application prospect.

Inventors

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Assignees

  • 杭州唯铂莱生物科技有限公司

Dates

Publication Date
20260508
Application Date
20230310

Claims (13)

  1. 1. A genetically engineered bacterium comprising genes encoding vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase, or The genetically engineered bacteria comprise genes for encoding O-demethylase, vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase; the O-demethylase has an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 2; The vanillyl alcohol oxidase has an amino acid sequence shown as SEQ ID NO.5 or SEQ ID NO. 6; The coniferyl alcohol dehydrogenase has an amino acid sequence shown as SEQ ID NO.9 or SEQ ID NO. 10; The coniferyl aldehyde dehydrogenase has an amino acid sequence shown as SEQ ID NO.13 or SEQ ID NO. 14; the host of the genetically engineered bacterium is escherichia coli.
  2. 2. The genetically engineered bacterium of claim 1, wherein the O-demethylase has a nucleotide sequence set forth in seq id No.3 or seq id No. 4.
  3. 3. The genetically engineered bacterium of claim 1, wherein the vanillyl alcohol oxidase has a nucleotide sequence set forth in seq id No.7 or seq id No. 8.
  4. 4. The genetically engineered bacterium of claim 1, wherein the coniferyl alcohol dehydrogenase has a nucleotide sequence of seq id No.11 or seq id No. 12.
  5. 5. The genetically engineered bacterium of claim 1, wherein the coniferyl aldehyde dehydrogenase has a nucleotide sequence of seq id No.15 or seq id No. 16.
  6. 6. The genetically engineered bacterium of claim 1, wherein the escherichia coli is selected from any one of ESCHERICHIA COLIBL (DE 3), ESCHERICHIA COLIDH5 a, and ESCHERICHIA COLI XL-Blue.
  7. 7. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium comprises an expression vector comprising a pcdfdurt-1 plasmid and a pACYCDuet-1 plasmid.
  8. 8. The method of constructing a genetically engineered bacterium of any one of claims 1 to 7, wherein the method of constructing is selected from any one of (1) or (2) below: (1) Connecting genes of vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase to an expression vector, and then introducing the obtained recombinant expression vector into the escherichia coli to obtain the genetically engineered bacterium; (2) And connecting genes of the O-demethylase, the vanillyl alcohol oxidase, the coniferyl alcohol dehydrogenase and the coniferyl aldehyde dehydrogenase to an expression vector, and then introducing the obtained recombinant expression vector into the escherichia coli to obtain the genetically engineered bacterium.
  9. 9. A whole-cell catalyst comprising the genetically engineered bacterium of any one of claims 1 to 7.
  10. 10. The use of the genetically engineered bacterium of any one of claims 1-7 for the production of cinnamic acid and its derivatives, wherein the derivatives are 3, 4-dihydroxycinnamic acid; The method for producing cinnamic acid by using the genetically engineered bacterium of any one of claims 1 to 7 comprises the steps of adding the genetically engineered bacterium into a solution containing benzene and propylene for whole cell transformation to obtain a target product, wherein the genetically engineered bacterium comprises genes for encoding vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase; The method for producing 3, 4-dihydroxycinnamic acid by using the genetically engineered bacterium of any one of claims 1 to 7 comprises the steps of adding the genetically engineered bacterium into a eugenol-containing solution for whole cell transformation to obtain a target product, wherein the genetically engineered bacterium comprises genes for encoding O-demethylase, vanillyl alcohol oxidase, coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase.
  11. 11. The use according to claim 10, wherein the whole cell transformed production system for producing cinnamic acid comprises 1-15g/L of phenylpropene, 5-30g/L of glucose and 1-20g/L of genetically engineered bacteria; The whole cell transformation production system for producing 3, 4-dihydroxycinnamic acid comprises eugenol 1-15g/L, glucose 5-30g/L and genetically engineered bacteria 1-20g/L.
  12. 12. The use according to claim 11, characterized in that the temperature of the whole cell transformed production system is 15-40 ℃.
  13. 13. The use according to claim 11, wherein the whole cell transformed production system has a reaction time of 10-24 h.

Description

Genetically engineered bacterium for producing cinnamic acid and derivatives thereof, construction method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a genetically engineered bacterium for producing cinnamic acid and derivatives thereof, a construction method and application thereof. Background Cinnamic acid, also known as beta-phenylacrylic acid, 3-phenyl-2-acrylic acid, is an organic substance with a chemical formula of C 9H8O2, and is an organic acid separated from cortex Cinnamomi or benzoin. The phenylalanine produced by deamination degradation of phenylalanine in plants. Cinnamic acid is mainly used in the aspects of essence and spice, food additive, medical industry, cosmetology, pesticide, organic synthesis, etc. The 3, 4-dihydroxycinnamic acid, also called caffeic acid, widely exists in various plants, can be used as a natural antioxidant, is widely applied to industries such as cosmetics, food and medicines, and the like, is an important precursor compound for synthesizing caffeic acid derivatives, and can be used for synthesizing various products with high added values such as chlorogenic acid, caffeic acid phenethyl ester and the like. The preparation method of cinnamic acid and its derivatives (such as caffeic acid) mainly comprises extraction method, chemical synthesis method and bioconversion method. The extraction method mainly uses natural plants as raw materials, and the caffeic acid is obtained by extraction, soaking and other steps, and the method has high cost, low yield and low purity, so that the industrialized application of the method is limited. The chemical synthesis method has the defects of more byproducts, difficult purification, difficult control of reaction conditions and the like, and is not suitable for large-scale industrial production. The existing biosynthesis route of cinnamic acid and its derivatives has the disadvantages of low yield, low production efficiency, high substrate cost, long reaction period, etc., for example, china patent CN106701843 takes catechol, pyruvic acid and ammonia as substrates, and generates caffeic acid under the action of tyrosine benzene lyase and tyrosine amino lyase, and the maximum caffeic acid yield finally obtained is only 35.5mg/L. Therefore, the method for preparing the cinnamic acid and the derivative thereof has higher yield, high production efficiency and low cost and has higher application value. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a genetic engineering bacterium for producing cinnamic acid and derivatives thereof, a construction method and application thereof, and the genetic engineering bacterium is used as a biocatalyst to convert a substrate into the cinnamic acid and derivatives thereof. The method has the advantages of high production efficiency, short reaction period, high target product yield, low cost and good industrial application prospect. The invention provides a new preparation route for overcoming the defects of low yield, low production efficiency, high substrate cost, long reaction period and the like of cinnamic acid and derivatives thereof in the prior art. Aiming at cinnamic acid, the invention takes the phenylpropene as a raw material, converts the phenylpropene into the cinnamyl alcohol through vanillyl alcohol oxidase, converts the cinnamyl alcohol into the cinnamyl aldehyde through coniferyl alcohol dehydrogenase, generates the cinnamic acid under the action of coniferyl aldehyde dehydrogenase, and requires coenzyme in the reaction process, and the reaction principle is shown in figure 1. Of course, any enzyme capable of converting styrene into cinnamyl alcohol or an enzyme capable of converting cinnamyl alcohol into cinnamyl aldehyde or an enzyme capable of converting cinnamyl aldehyde into cinnamic acid is suitable for the present invention, for example, alcohol dehydrogenase (EC1.1.1.1) or cinnamyl alcohol dehydrogenase (EC1.1.1.195) may replace CalA, acetaldehyde dehydrogenase (EC1.2.1.3) or acetaldehyde oxidase (EC1.2.3.1) may replace CalB, and the like, and the present invention does not list such enzymes one by one. The present invention is not limited to a specific enzyme. Aiming at 3, 4-dihydroxycinnamic acid, the invention takes eugenol as a raw material, converts the eugenol into 4-allylcatechol by O-demethylase, converts the 4-allylcatechol into 3, 4-dihydroxycinnamic alcohol by vanillyl alcohol oxidase, converts the 3, 4-dihydroxycinnamic alcohol into 3, 4-dihydroxycinnamic aldehyde by coniferyl alcohol dehydrogenase, generates the 3, 4-dihydroxycinnamic acid under the action of coniferyl aldehyde dehydrogenase, requires coenzyme in the reaction process, and the reaction principle is shown in figure 2. Of course, any enzyme capable of converting eugenol into 4-allylcatechol or capable of converting 4-allylcatechol into 3, 4-dihydroxycinnamyl alcohol or ca