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A kind of engineering bacterium and its application in producing l-tyrosine

The technology of tyrosine and tyrosine phenol is applied in the field of bioengineering, which can solve the problems of easy decomposition of pyruvic acid and low efficiency, and achieve the effects of high NAD content, simple production process and good industrial application prospect.

Active Publication Date: 2020-12-29
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 solution to directly convert, or first oxidize lactic acid with lactic acid oxidase to generate pyruvic acid, and then further catalyze the production of L-tyrosine (multiple Enzyme-coupled biosynthesis of pyruvate and L--tyrosine, 2014, Master 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 bacterium and its application in producing l-tyrosine
  • A kind of engineering bacterium and its application in producing l-tyrosine

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Experimental program
Comparison scheme
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, 1 g / L of phenol or L-tyrosine, 100 g / L of wet bacteria, and...

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, phenol 10g / L, pH 7.0, temperature 30°C, shaker speed 250 rpm; transformation time 10 hours.

[0076] The various expression plasmids of table 3 are for the comparison of producing L-tyrosine

[0077] strain L-Tyrosineg / L Escherichia coli HM / pETDuet-1-llnox-llldh-cftpl 6.3 Escherichia coli HM / pACYCDuet-1-llnox-llldh-cftpl 4.3 Escherichia coli HM / pCOLADuet-1-llnox-llldh-cftpl 5.7 Escherichia coli HM / pRSFDuet-1-llnox-llldh-cftpl 5.3 Escherichia coli HM / pCDFDuet-1-llnox-llldh-cftpl 4.8 ...

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Abstract

The invention discloses an engineering bacterium, and an application thereof in the production of L-tyrosine, and belongs to the technical field of bioengineering. The invention also provides a production method for converting lactic acid and phenol to produce L-tyrosine without generating hydrogen oxide. The three-enzyme co-expression engineering bacterium is constructed on the basis of reconstructing Escherichia coli transportation and a coenzyme synthesis system in order to realize the efficient production of the L-tyrosine. The method for whole cell transformation to produce L-tyrosine hasthe advantages of simple process, few impurities and great industrial application values.

Description

technical field [0001] The invention relates to an engineering bacterium and its application in producing L-tyrosine, belonging to the technical field of bioengineering. Background technique [0002] L-tyrosine (p-hydroxyphenylalanine) is an essential amino acid for the human body and has important applications in medicine and food. At present, L-tyrosine is extracted, enzymatically, and fermented by genetically engineered bacteria. The process of hydrolyzing and extracting L-tyrosine from wool method and other raw materials has a lot of pollution and low purity. Escherichia coli engineering bacteria use glucose as a raw material to synthesize L-tyrosine with a low yield, which is currently unable to compete with extraction and enzymatic methods. [0003] Enzymatic production of L-tyrosine with tyrosine phenol lyase is the most widely used method. A variety of schemes have been designed (Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/22C12R1/19
CPCC12N9/0036C12N9/88C12P13/225C12Y101/01C12Y101/01027C12Y106/99003C12Y401/99002
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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