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A kind of engineering bacteria and its application in producing caffeic acid

A technology of caffeic acid and strain, applied in the field of bioengineering, can solve the problems of easy decomposition, low yield of caffeic acid, high price of dopa, etc.

Active Publication Date: 2022-08-09
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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  • A kind of engineering bacteria and its application in producing caffeic acid
  • A kind of engineering bacteria and its application in producing 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 single-sampled or double knockout. Wherein, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and homology arm (hpaDdonor) together into Escherichia coli BL21 (DE3), Cas9 / sgRNA induces the host to produce double-stranded at the hpaD gene site Fragmentation, recombinase Red integrates the hpaD donor into the hpaD gene to achieve gene knockout, and sequence verification. hpaD sgRNA, hpaD donor, mhpB sgRNA, mhpB donor are shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 in the sequence table, respectively. mhpB was knocked out in the same way.

[0066] A solution with a pH of 8, 2 g / L of catechol or caffeic acid, 100 g / L of wet bacterial mass, was placed at 35...

Embodiment 2

[0071] Construction of recombinant Escherichia coli: First, the genes encoding tyrosine phenol lyase, tyrosine ammonia lyase, NADH oxidase and L-lactate dehydrogenase were ligated into pETDuet-1 or pACYCDuet-1 plasmids, respectively. Two 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] Induction expression method: The recombinant Escherichia coli was transferred to LB fermentation medium (10g / L of peptone, 5g / L of yeast powder, 10g / L of NaCl) according to the volume ratio of 2%. 600 After reaching 0.6-0.8, IPTG with a final concentration of 0.4 mM was added to induce expression and culture at 20°C for 8 h. After induction of expression, cells were collected by centrifugation at 20°C, 8000 rpm, and 20 minutes.

[0073] After the recombinant Esch...

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 genes on the Escherichia coli HM genome were increased in front of Escherichia coli A moderate expression strength constitutive promoter (PG) in front of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), the sequence is shown in SEQ ID NO:9.

[0078] When the expression of the gene lldP ​​was enhanced, the Escherichia coli HM genome was used as the template, and the primers lldP-FF / lldP-FR, lldP-gpdA-F / lldP-gpdA-R, lldP-RF / lldP-RR were used to amplify the upstream and start 11dP-FF and lldP-RR were used as primers to fuse into an expression cassette containing the gpdA promoter. Then, together with plasmid pCasRed and pCRISPR-gDNA (containing lldP ​​sgRNA) into Escherichia coli HM, Cas9 / sgRNA induces the host to have double-strand breaks at the lldP ​​gene ...

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Abstract

The invention discloses an engineering bacterium and its application for producing caffeic acid, and belongs to the technical field of biological engineering. The invention provides a recombinant bacteria capable of producing caffeic acid at low cost; the recombinant bacteria simultaneously expresses four enzymes, namely tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase, and NADH oxidase; Further, the recombinant bacteria of the present invention also knocks out the phenolic substance decomposition gene, and strengthens the expression of any one or more of the lactic acid transport gene, the catechol transport gene, and the coenzyme synthesis-related gene. The invention realizes the efficient production of caffeic acid, and has the advantages of simple process and few impurities, and has important industrial application value.

Description

technical field [0001] The invention relates to an engineering bacterium and its application for producing caffeic acid, and belongs to the technical field of biological engineering. Background technique [0002] Caffeic acid is a phenolic acid compound widely present in various plants and an important pharmaceutical intermediate. At present, the main sources of caffeic acid are chemical synthesis and plant extraction, but they are expensive. Escherichia coli engineering bacteria use glucose as raw material for 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 the raw material is still very low, and the separation of impurities in the fermentation broth is difficult (US20150184205). The enzymatic conversion method usually uses dopa as a raw material and is converted by tyrosine ammonia lyase, but the high cost of dopa leads to high cost (CN20161...

Claims

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Application Information

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Patent Type & Authority Patents(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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