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A recombinant Corynebacterium glutamicum producing l-phenylalanine and its construction and application

A glutamic acid stick and phenylalanine technology, applied in the field of metabolic engineering, can solve the problems of Escherichia coli limitation and achieve the effect of increasing production

Inactive Publication Date: 2017-09-15
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although Escherichia coli can ferment and produce L-phenylalanine to a very high level, Escherichia coli is limited in large-scale industrial production due to its own characteristics, especially in the production of food-grade large-scale industrial production

Method used

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  • A recombinant Corynebacterium glutamicum producing l-phenylalanine and its construction and application
  • A recombinant Corynebacterium glutamicum producing l-phenylalanine and its construction and application
  • A recombinant Corynebacterium glutamicum producing l-phenylalanine and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of recombinant plasmids pSUTL and pSDTL, and construction of recombinant strain C. glutamicum (pSUTL, pSDTL).

[0049] Overexpression of four genes aroF with pEC-XK99E (GenBank: AY219683.1) fbr , aroE, ppsA and tktA, combined with two promoters Ptac and Plac at the same time to construct the recombinant plasmid pECXK99E-Ptac-aroF fbr -aroE-Plac-ppsA-tktA is abbreviated as pSUTL. Four genes pheA were overexpressed with pXMJ19 (Jakoby M, Ngouoto-Nkili CE, BurkovskiA. Construction and application of new Corynebacterium glutamicum vectors[J]. Biotechnology techniques.1999.13(6):437-441) fbr , aroA, tyrB and aroL, combined with two promoters Ptac and Plac at the same time to construct the recombinant plasmid pXMJ19-Ptac-pheA fbr -aroA-Plac-tyrB-aroL is abbreviated as pSDTL.

[0050] Schematic diagram of the construction process of the regulatory expression vectors pSUTL and pSDTL according to the construction figure 1 As shown, aroE, ppsA, tktA and...

Embodiment 2

[0061] Example 2 Effect of Overexpression of Eight Key Enzyme Genes of L-Phe Synthetic Pathway in Recombinant Corynebacterium glutamicum C. glutamicum (pSUTL, pSDTL) on L-Phe Fermentation

[0062] Seed activation medium (LBG) (g / L): peptone 10.0, yeast extract 5.0, sodium chloride 10.0, glucose 5.0. Liquid volume 20mL / 250mL.

[0063] Seed activation medium (LBG solid) (g / L): glucose 5.0, yeast powder 5.0, peptone 10.0, sodium chloride 10.0, nutrient agar 15.0-20.0.

[0064] Fermented seed medium (g / L): glucose 25.0, corn steep liquor dry powder 17.5, ammonium sulfate 5.0, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, urea 2.0, pH 6.8-7.0. Liquid volume 20mL / 250mL.

[0065] Fermentation medium (g / L): glucose 100.0, corn steep liquor dry powder 6.0, ammonium sulfate 25.0, magnesium sulfate 0.5, potassium dihydrogen phosphate 1.0, sodium citrate 2.0, calcium carbonate 20.0, pH 6.8-7.0. Liquid volume 20mL / 250mL.

[0066] Corynebacterium glutamicum medium was added...

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Abstract

The invention discloses a recombined Corynebacterium glutamicum for producing L-Phe and a constructing method and the application thereof, and belongs to the field of metabolic engineering. In Corynebacterium glutamicum ATCC 13032, two shuttle expression vectors pEC-XK99E and pXMJ19 of the Corynebacterium glutamicum and escherichia coli are used for expressing eight key enzyme genes comprising aroF<fbr>, tktA, ppsA, aroL, pheA<fbr>, aroE, aroA and tyrB in a L-Phe synthetic route, two promoters Ptac and Plac of different intensities are used for carrying out combinational expression on the eight genes to improve the L-Phe yield, the L-Phe yield can reach as high as 5.59+ / -0.11 g / l, and accumulated shikimic acid is 0.31+ / -0.11 g / L. According to the method, over expression is achieved for the L-Phe shikimic acid key enzyme genes, and the yield of L-Phe produced by the Corynebacterium glutamicum in a fermentation mode is improved.

Description

technical field [0001] The invention relates to a recombinant corynebacterium glutamicum producing L-phenylalanine and its construction and application, belonging to the field of metabolic engineering. Background technique [0002] Phenylalanine (Phe), namely D.L-α-amino-β-phenylpropionic acid, is an aromatic heterocyclic, non-polar, electrically neutral amino acid with racemic DL-type, There are three kinds of L-type and D-type, the optical isomer with biological activity is L-phenylalanine (L-Phe), and the specific rotation is -35.1°. L-Phe can be irreversibly converted into L-tyrosine (L-Tyrosine, L-Tyr) by the coenzyme tetrahydrobiopterin in the living body, and then continue to decompose and generate a small amount of phenylpyruvate through transamination. L-Phe widely exists in nature and is one of the eight essential amino acids for human and animals, while D-Phe does not exist in nature and can only be obtained through synthetic methods. [0003] The production tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/22C12R1/15
CPCC12N9/0006C12N9/1022C12N9/1085C12N9/1092C12N9/1096C12N9/1205C12N9/88C12N15/77C12P13/222C12Y101/01025C12Y202/01001C12Y205/01019C12Y207/01071C12Y402/01051
Inventor 陈坚堵国成康振张传志顾汉章徐堃
Owner JIANGNAN UNIV
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