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A kind of engineering bacteria and its application in the 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: 2021-03-26
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • A kind of engineering bacteria and its application in the production of levodopa
  • A kind of engineering bacteria and its application in the production of levodopa
  • A kind of engineering bacteria and its application in the 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 singled out Or double knockout, wherein, the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) and homologous arm (hpaDdonor) are introduced together on Escherichia coli BL21 (DE3), Cas9 / sgRNA induces host in hpaD gene A double-strand break occurred at the site, and the recombinase Red integrated the hpaD donor into the hpaD gene to achieve gene knockout, which was verified by sequencing. hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor are respectively shown in the sequence listing SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. mhpB was knocked out in the same way.

[0063] Prepare a solution with a pH of 8, catechol or levodopa 2g / L, wet bacterial mass 100g / L, and me...

Embodiment 2

[0068] Recombinant Escherichia coli construction: First, the genes encoding lactate dehydrogenase, NADH oxidase, and tyrosine phenolic acid lyase were respectively connected to the 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] 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.

[0070] The collected cells were analyzed for transformation, and the results are shown in ...

Embodiment 3

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

[0076] The comparison of various expression plasmids of table 3 for producing levodopa

[0077]

[0078]

[0079] It can be seen from the above table that the effect is the best when pETDuet-1 is used for co-expression.

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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 producing levodopa, belonging to the technical field of bioengineering. Background technique [0002] Levodopa (levodopa, L-DOPA, 3-hydroxy-L-tyrosine), is an important compound used in the treatment of Kimson's disease. At present, sources of levodopa include cat bean extraction method, chemical synthesis method, enzymatic method, and genetically engineered bacteria. The content of L-DOPA in cat bean can reach more than 9%, but it is greatly affected by planting area, climate, etc., and the output is limited (extraction and content determination of L-DOPA in cat bean shell, natural product research and development, 1992,4 (4): 27-30.Chemical synthesis method needs to use a large amount of metal catalysts, can produce bigger pollution (US Pat. Engineering Escherichia Coli for L-DopaOverproduction from Glucose.Scientific Reports, 2016.6:30080), currently unable to compete with ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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