Engineering bacterium, and application thereof in production of caffeic acid

A technology of caffeic acid and recombinant bacteria, applied in the field of bioengineering, can solve the problems of high cost, easy decomposition, low yield of caffeic acid, etc., and achieve the effects of simple production process, easy availability of raw materials, and good prospects for industrial application.

Active Publication Date: 2018-12-07
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

[0003] The yield of caffeic acid produced by the fermentation method using glucose as raw material is still very low, and there are many impurities in the fermentation broth and it is difficult to separate them (US20150184205)
The enzymatic conversion method usually uses dopa as a raw material and is converted by tyrosine ammonia lyase, but dopa is expensive and leads to higher costs (CN201611158372.2); or co-express tyrosine phenol cleavage and tyrosine ammonia cleavage The enzyme synthesizes dopa with pyruvate, catechol and ammonia as substrates, and then converts it into caffeic acid with intracellular tyrosine ammonia lyase (CN201611166591.5), but the price of pyruvate is relatively high and it is easy to decompose

Method used

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  • Engineering bacterium, and application thereof in production of caffeic acid
  • Engineering bacterium, and application thereof in production of caffeic acid

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Experimental program
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Effect test

Embodiment 1

[0065] According to the method described in the literature Large scale validation of an efficient CRISPR / Cas-based multigene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1): 68, hpaD and mhpB on Escherichia coli BL21 (DE3) were singled out or double knockout. Among them, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and the homology arm (hpaDdonor) are introduced into Escherichia coli BL21 (DE3), and Cas9 / sgRNA induces the host to generate double strands at the hpaD gene locus Break, the recombinase Red integrates the hpaD donor into the hpaD gene to achieve gene knockout, and sequence verification. hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor are respectively shown in the sequence listing SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. mhpB was knocked out in the same way.

[0066] Prepare a solution with a pH of 8, catechol or caffeic acid 2g / L, wet bacterial mass 100g / L, and m...

Embodiment 2

[0071] Recombinant Escherichia coli construction: Firstly, the genes encoding tyrosine phenol lyase, tyrosine ammonia lyase, NADH oxidase and L-lactate dehydrogenase were connected to pETDuet-1 or pACYCDuet-1 plasmid respectively. Two kinds of double-gene co-expression recombinant plasmids were obtained, and the two plasmids were transformed into Escherichia coli HM, and positive transformants were obtained by screening with chloramphenicol and ampicillin plates, that is, recombinant Escherichia coli was obtained.

[0072] Induced expression method: the recombinant Escherichia coli is transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in the amount of 2% by volume ratio, when the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.4mM, induce expression and culture at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 20° C., 8000 rpm, and 20 minutes.

[0073] After the induction...

Embodiment 3

[0077]Using the method described in the literature Large scale validation of an efficient CRISPR / Cas-based multigene editing protocol in Escherichia coli.Microbial Cell Factories, 2017,16(1):68, the corresponding gene on the Escherichia coli HM genome was increased before the Escherichia coli The moderate expression strength constitutive promoter (PG) in front of the 3-phosphate glyceraldehyde dehydrogenase gene (gpdA), the sequence is shown in SEQ ID NO:9.

[0078] When enhancing the expression of the gene lldP, use the Escherichia coli HM genome as a template, and use the primers lldP-FF / lldP-FR, lldP-gpdA-F / lldP-gpdA-R, lldP-RF / lldP-RR to amplify the upstream, promoter sub, downstream sequences, and lldP-FF and lldP-RR primers were fused into an expression cassette containing the gpdA promoter. Then, after the plasmid pCasRed and pCRISPR-gDNA (including lldP ​​sgRNA) were transferred into Escherichia coli HM, Cas9 / sgRNA induced the host to undergo a double-strand break at t...

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Abstract

The invention discloses an engineering bacterium, and an application thereof in the production of caffeic acid, and belongs to the technical field of bioengineering. The engineering bacterium providedby the invention is a recombinant bacterium capable of producing caffeic acid at a low cost; the recombinant bacterium can simultaneously express four enzymes which are tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase respectively; and the recombinant bacterium knocks out a phenolic substance-decomposing gene, and can achieve enhanced expression of any one or more of a lactic acid transporter gene, a catechol transporter gene and a coenzyme synthesis-related gene. The engineering bacterium has the advantages of realization of the efficient production ofcaffeic acid, simple process, few impurities and great industrial application values.

Description

technical field [0001] The invention relates to an engineering bacterium and its application for producing caffeic acid, belonging to the technical field of bioengineering. Background technique [0002] Caffeic acid is a phenolic acid compound widely present in various plants and is an important pharmaceutical intermediate. At present, the main sources of caffeic acid are chemical synthesis and plant extraction, but they are all expensive. Escherichia coli engineering bacteria use glucose as raw material to de novo synthesis or enzyme-catalyzed precursor synthesis is the focus of current research. [0003] The yield of caffeic acid produced by the fermentation method using glucose as raw material is still very low, and there are many impurities in the fermentation broth and it is difficult to separate (US20150184205). The enzymatic conversion method usually uses dopa as a raw material and is converted by tyrosine ammonia lyase, but dopa is expensive and leads to higher cos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/42C12R1/19
CPCC12N9/0006C12N9/0036C12N9/88C12P7/42C12Y101/01027C12Y106/99003C12Y401/99002C12Y403/01023
Inventor 蔡宇杰熊天真蒋静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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