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ARO3 protein mutant and application thereof

A mutant and protein technology, applied in ARO3 protein mutants and their application fields, can solve the problem of not finding mutants

Active Publication Date: 2021-07-27
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ARO3 has not yet found a mutant that can alleviate the feedback inhibition of phenylalanine

Method used

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  • ARO3 protein mutant and application thereof
  • ARO3 protein mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 ARO3 D154N Outer application of mutant protein and related plasmids, strains

[0041] Will encode ARO3 D154N The gene of the mutant (SEQ ID NO: 3) is inserted into the Escherichia coli expression vector PET21b to obtain a recombinant carrier.

[0042] The above recombinant vectors were converted to the expression of E. coli Rosetta (DE3).

[0043] The above strains were then tedped overnight, then transferred to Lb medium (100 μg / ml ampiconbin), and raised to OD. 600 = 0.6-0.8, the final concentration was 0.1 mmIPTG, 16 ° C, 110 rpm overnight induced expression. After collecting bacteria, centrifugation was collected after the breast, and then the mutant protein was collected using a phosphate buffer with low imidazole (20 mm imidazole) after incubation with nickel gel. The mutant protein was collected by a phosphate buffer with 250 mM imidazole. Phosphate buffer: 50 mM potassium hydrogen phosphate, pH is 6.5-6.8, 150 mM sodium chloride. The eluted protein dialy...

Embodiment 2

[0046] Example 2 ARO3 D154N Application of mutant protein and related plasmids, strains

[0047] 2.1 Sacchal yeast genome original position mutation ARO3 is ARO3 D154N

[0048] On the primary strain Cen.PK2-1 C strain genome, CRISPR / CAS9-mediated gene settling point mutation technique; the method of genetically fixed-point mutation, the plasmid of the plasmid carrying the Cas9 protein gene and a gene used to transcribe, transcribed RNA Combined with CAS9 protein, simply introduce a gene Spacer and homologous recombinant fragments that match the target gene on the plasmid, and will refer to the product into the cell, because the homologous recombinant fragment is fixed, and the yeast is eugenic The target gene in which the cell was cut, in homologous recombination, the homogeneous arm on the plasmid was a template, and the fixed-point mutation was completed, thereby completing the purpose of the genetically fixed point mutation.

[0049] Spacer: AcccattgctggTGAGATGT

[0050] Hom...

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Abstract

The invention relates to the technical field of biology, in particular to an ARO3 protein mutant and an application thereof. The ARO3 mutant disclosed by the invention is obtained by mutating the 154th amino acid of ARO3 protein in saccharomycetes from aspartic acid to asparagine. The experiments prove that the mutant ARO3D154N protein can relieve allosteric inhibition of phenylalanine on the ARO3 protein, the mutant protein is expressed in microorganisms, the biosynthesis capacity of microorganism aromatic amino acid derivatives is obviously enhanced, and the industrial application prospect is good.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to ARO3 protein mismaries and their applications. Background technique [0002] The aromatic amino acid derivative is an intermediate of many natural products and pharmaceuticals. At the same time, the aromatic amino acids and their derivatives are also widely used in the fields of health products, drugs, and cosmetics, have extensive application prospects and development value. Microbial fermentation produces aromatic amino acids and its derivatives have advantages of less dependence on natural environments and low prices. The rocky acid pathway and the immediate aromatic amino acid pathway are key ways for Saccharomyces to synthesize aromatic amino acids and their derivatives. [0003] The starting step of the glutinous acid pathway in Saccharomyces is Erythoxo-4-phosphate (E4P) and phosphoenolpyruvate, PEP) to form DAHP under the action of DAHP synthase, ie 3-deoxidation-α-Arabic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/81C12N1/19C12P13/04C12R1/865
CPCC12N9/00C12N15/81C12P13/04
Inventor 罗云孜刘化一
Owner TIANJIN UNIV
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