Genetically engineered bacterium for producing ferulic acid and biosynthesis method of ferulic acid

A genetically engineered bacteria and biosynthesis technology, applied in the field of biological applications, can solve the problem of high production cost, and achieve the effects of low cost, simple and clean process, and short process flow

Inactive Publication Date: 2020-09-25
SICHUAN INGIA BIOSYNTHETIC CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method adopts the in vivo conversion method of genetic engineering enzymes, which saves the extraction process of enzymes, but still uses relatively expensive caffeic acid as raw material, and the production cost is high.

Method used

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  • Genetically engineered bacterium for producing ferulic acid and biosynthesis method of ferulic acid
  • Genetically engineered bacterium for producing ferulic acid and biosynthesis method of ferulic acid
  • Genetically engineered bacterium for producing ferulic acid and biosynthesis method of ferulic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The construction of embodiment 1 genetically engineered bacteria

[0060] Referring to the biosynthetic pathway of ferulic acid in plants, and according to the protein sequence reported in NCBI, the codon optimization of the gene sequence was carried out to adapt it to the E. coli expression system. Artificially synthesized gene fragments of tyrosine ammonia lyase (TAL), S-adenosylmethionine synthetase 5 (Sam5), and caffeic acid-3-O-methyltransferase (COMT) were sequentially ligated into pBR322 or pET21d vector, and transformed into JM109, BL21(DE3) or Rosetta competent cells, and positive clones were screened. The recombinant strains screened out were subjected to enzyme digestion verification, and sequenced to confirm that the sequence was correct, and the expected production strain was obtained. Specifically, the following methods are used:

[0061] According to the protein sequences of TAL, Sam5 and COMT, the codons were optimized, and three genes were artificiall...

Embodiment 2

[0063] Embodiment 2 Shake flask conversion produces ferulic acid

[0064] After thawing the strains prepared by the method of Example 1 stored at -80°C, they were streaked on LB agar plates and cultured overnight at 37°C. Pick a single clone, inoculate in 20mL LB broth medium containing antibiotics, and culture in a 250mL Erlenmeyer flask at 37°C and 200rpm for 12h to obtain seed liquid. Take another 5 mL and inoculate in 100 mL M9 medium (ammonium chloride 1 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 3 g / L, yeast extract 0.5 g / L, magnesium sulfate 2 mM, calcium chloride 0.1 mM , glycerol 20g / L, L-tyrosine 1g / L, L-methionine 1g / L, IPTG 0.1mM, antibiotic 100mg / L), 25 ℃ 200rpm cultivated for 3 days, obtained the fermented liquid containing ferulic acid.

[0065] The conversion rate was determined as follows:

[0066] Extract 5-10 mL of fermentation broth with an equal volume of ethyl acetate three times, combine the extracts, concentrate and dry, th...

Embodiment 3

[0070] Embodiment 3 fermentation tank produces ferulic acid

[0071] After the FA-J strains prepared by the method in Example 1 were thawed, they were streaked on an agar plate and cultured overnight at 37°C. Single clones were picked, inoculated in 150 mL LB broth medium, and cultured at 37°C for 10 h. Then inoculated in 3L TB medium (24g / L yeast extract, 12g / L peptone, 2.31g / L sodium dihydrogen phosphate, 17.4g / L disodium hydrogen phosphate, 20g / L glucose, 3g L-tyrosine / L, L-methionine 3g / L, IPTG 0.1mM, antibiotic 100mg / L, feeding 400g / L glucose) in a 5L fermenter, cultivated at 28°C, pH control 7.0, ventilation 3L / min, controlled dissolved oxygen It is not lower than 30%, the tank pressure is not more than 0.07MPa, the dissolved oxygen is controlled by the rotating speed and feeding speed, and the tank is put into the tank after culturing for 60 hours to obtain a ferulic acid-containing fermented liquid. The conversion results are shown in Table 2.

[0072] Table 2

[...

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Abstract

The invention relates to a genetically engineered bacterium for producing ferulic acid and a biosynthesis method of the ferulic acid, and belongs to the technical field of biological application. Thegenetically engineered bacterium for producing the ferulic acid can produce the ferulic acid by using tyrosine and methionine as raw materials. According to the biosynthesis way of the ferulic acid inplants, the recombinant genetically engineered bacterium is obtained by a genetic engineering method, and the genetically engineered bacterium can heterologously express tyrosine ammonia lyase, coumaric acid-3-hydroxylase and caffeic acid-3-O-methyltransferase, so that tyrosine biotransformation is promoted, the ferulic acid is produced by one-pot fermentation, the conversion rate is high, the purity of the obtained ferulic acid is high, the process is simple and clean, the cost is low, and the genetically engineered bacterium for producing the ferulic acid and the biosynthesis method of theferulic acid can be used for large-scale industrial production.

Description

technical field [0001] The invention relates to a gene engineering bacterium for producing ferulic acid and a biosynthetic method of ferulic acid, belonging to the technical field of biological applications. Background technique [0002] Ferulic acid, the chemical name is 3-methoxy-4-hydroxycinnamic acid, which belongs to the derivative of cinnamic acid. It is ubiquitous in plants, especially in Chinese medicinal materials such as Ferulicum, Chuanxiong, and Angelica. It is also one of the active ingredients of these Chinese herbal medicines. The content of ferulic acid is also high in food raw materials such as coffee, chaff, vanilla beans, wheat bran, and rice bran. [0003] Ferulic acid is divided into two configurations, cis and trans, the cis is a yellow oil, and the trans is a square crystal or fiber crystal. Among them, trans-ferulic acid can scavenge free radicals, promote the production of enzymes that scavenge free radicals, increase the activity of glutathione su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/60C12N15/53C12N15/70C12P7/42C07C51/42C07C51/47C07C51/48C07C59/64C12R1/19
CPCC07C51/42C07C51/47C07C51/48C12N9/1007C12N9/1085C12N9/88C12N15/70C12P7/42C12Y201/01068C12Y205/01006C12Y403/01023C07C59/64
Inventor 华君王勇裴亮潘月华卿汝
Owner SICHUAN INGIA BIOSYNTHETIC CO LTD
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