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Engineering bacterium, and application thereof in production of levodopa

A technology for recombining Escherichia coli and genes, applied in the field of bioengineering, can solve problems such as low efficiency and easy decomposition of pyruvate, 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
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AI Technical Summary

Problems solved by technology

Pyruvate is a relatively expensive intermediate, so patent 201310289373.0 uses unpurified pyruvic acid feed liquid to directly convert, or first oxidize lactic acid with lactic acid oxidase to generate pyruvic acid, and then further catalyze the production of levodopa (or tyrosine acid) (multi-enzyme coupling biosynthesis of pyruvate and L--tyrosine, 2014, master's thesis of Nanjing University), but pyruvate is easy to decompose, and the efficiency of this scheme is not high

Method used

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  • Engineering bacterium, and application thereof in production of levodopa
  • Engineering bacterium, and application thereof in production of levodopa
  • Engineering bacterium, and application thereof in production of levodopa

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Experimental program
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Effect test

Embodiment 1

[0062]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 plasmid of the gene knockout 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 host in hpaD gene A double-strand break occurs at the site, and the 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 in the sequence table, respectively. mhpB was knocked out in the same way.

[0063] A solution with a pH of 8, 2g / L of catechol or levodopa, 100g / L of wet bacterial mass, was placed at 35°C for ...

Embodiment 2

[0068] Construction of recombinant Escherichia coli: Firstly, the genes encoding lactate dehydrogenase, NADH oxidase and tyrosine phenolic acid lyase were respectively connected to plasmid pETDuet-1. After obtaining various three-gene co-expression recombinant plasmids, the plasmids were transformed into Escherichia coli HM, and positive transformants were obtained by screening with ampicillin plate, that is, recombinant Escherichia coli was obtained.

[0069] Induction expression method: The recombinant Escherichia coli was transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in a 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.

[0070] The collected cells were subjected to transformation analysis and the results are shown in Table 2. In t...

Embodiment 3

[0075] According to the strain construction method described in Example 2 (all types of plasmids use different resistance plates to screen positive transformants according to the instructions) and the induction expression method, various types of cells were collected for transformation analysis, and the results are shown in Table 3. In the transformation system, the whole cell transformation system is as follows: cell wet weight 50 g / L, L-lactic acid 10 g / L, catechol 10 g / L, pH 7.0, temperature 30 °C, shaking table rotation speed 250 rpm; transformation time 10 Hour.

[0076] Table 3 Comparison of various expression plasmids for the production of L-dopa

[0077]

[0078]

[0079] It can be seen from the above table that the co-expression of pETDuet-1 has the best effect.

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Abstract

The invention discloses an engineering bacterium, and an application thereof in the production of levodopa, and belongs to the technical field of bioengineering. The engineering bacterium is a recombinant Escherichia coli capable of producing pure levodopa at low cost; the recombinant Escherichia coli simultaneously expresses exogenous L-lactate dehydrogenase, NADH oxidase and tyrosine phenol lyase, and is obtained by knocking out an aromatic compound-degrading gene from host Escherichia coli; and the recombinant Escherichia coli can achieve enhanced expression of any one or more of a lactic acid transporter gene, an ammonia ion transporter gene, a catechol transporter gene, an NAD synthesis gene and a pyridoxal phosphate synthesis gene. A bacterium can be applied to the production of levodopa, and a method for producing the levodopa has the advantages of simple production process, few impurities, easily available raw materials and good industrial application prospect.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in the production of levodopa, and belongs to the technical field of biological engineering. Background technique [0002] Levodopa (levodopa, L-DOPA, 3-hydroxy-L-tyrosine), is an important compound for the treatment of Kinson's disease. At present, the sources of levodopa include cat bean extraction method, chemical synthesis method, enzymatic method and genetic engineering bacteria. The content of L-DOPA in cat beans can reach more than 9%, but it is greatly affected by planting area, climate, etc., and the yield is limited (extraction and content determination of L-DOPA in cat bean pod shells, Research and Development of Natural Products, 1992,4 (4): 27-30.Chemical synthesis method needs to use a large amount of metal catalysts, can produce larger pollution (U.S. Pat. No. 4,716,246).Escherichia coli engineering bacteria utilizes glucose to be that the productive rate of re-synthe...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/22C12R1/19
CPCC12N9/0006C12N9/0036C12N9/88C12P13/225C12Y101/01027C12Y106/99003C12Y401/99002
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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