High enzyme activity aspartokinase mutant, engineering bacterial strain and preparation method of mutant

A technology of aspartokinase and mutants, applied in the field of bioengineering, can solve problems such as heavy tasks, difficulty in further increasing target product yield, and long cycle time

Active Publication Date: 2019-02-01
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditionally, mutant strains are constructed by random mutation and subsequent screening through the action of physical or chemical mutagens, such as screening resistance to structural analogs and auxotrophic mutant strains. However, the above methods have heavy tasks, long periods and are difficult to further improve the target. production volume etc.

Method used

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  • High enzyme activity aspartokinase mutant, engineering bacterial strain and preparation method of mutant
  • High enzyme activity aspartokinase mutant, engineering bacterial strain and preparation method of mutant
  • High enzyme activity aspartokinase mutant, engineering bacterial strain and preparation method of mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1. Construction of recombinant E. coli strain

[0036] (1) Cloning of AK gene

[0037] The chromosomal DNA of Corynebacterium pekinense was extracted by TAKaRa genome extraction kit, and PCR amplification was carried out using it as a template under the action of cloning primers. A large number of AK target gene fragments were cloned. The PCR nucleic acid electrophoresis verified that Corynebacterium pekinense Acid kinase (AK) is attached figure 1 There is a bright band between 1000 and 2000 bp, which corresponds to the length and size of SEQ ID NO:1. The reagents were purchased from TAKaRa Company, and the designed cloning primers are as follows:

[0038] Upstream primer: 5'-GGAATTC CATATG GCCCTGGTCGTACAGAA-3'

[0039] Downstream primer: 5'-G GAATTC TTAGCGTCCGGTGCCTGCAT-3'

[0040] The underlined part is the restriction enzyme cut site of NdeI and EcoRI.

[0041]

[0042] (2) Construction of recombinant E. coli strain

[0043] The PCR product containing the AK target ...

Embodiment 2

[0059] Example 2. Construction of aspartate kinase mutants

[0060] (1) Construction of a mutant of aspartic kinase M372

[0061] Using the pET-AK recombinant plasmid in Example 1(2) as a template, the primer 5’-GTAACACCTGGGTGAGACTG upstream of M372N NNN GCCCGCACC-3’, downstream primer 5’-CTCGT GGGTGCGGGC NNN Carry out mutation PCR reaction under the action of CAGTCTCACCC-3' (the underlined part is the mutation site), and use restriction enzymes EcoRI and NdeI to double digest the PCR product and pET-28a vector and connect overnight (Using Example 1(2) Double restriction digestion and ligation system), the ligated product was transferred to E. coli BL21 (DE3) competent by the method 1(2) to obtain the aspartokinase M372 site mutant Strains.

[0062]

[0063]

[0064] (2) Construction of a mutant of aspartic kinase T379

[0065] Using the pET-AK recombinant plasmid in Example 1 (2) as a template, using the PCR reaction system and amplification program in Example 2 (1), the primer 5'-C...

Embodiment 3

[0074] Example 3. Enzyme kinetic analysis and characterization of enzyme properties

[0075] (1) Separation and purification of crude enzyme solution

[0076] The recombinant E. coli strain (WT) of Example 1 (2) and the high enzyme activity mutant (single mutant M372I and T379S) and double mutant M372I-T379S of Example 2 were inoculated at 2% (v / v) The amount was transferred to 100mL LB liquid medium containing kanamycin, after overnight culture at 37℃, 180r / min, IPTG inducer (final concentration 1mmol / L) was added, 30℃, 130r / min, induced 12h . Centrifuge the bacterial solution at 8000r / min for 10min, discard the supernatant, add 10mL PBS to resuspend the bacteria, sonicate for 30min, and centrifuge at 8000r / min for 10min. The supernatants obtained are WT crude enzyme solution, M372I crude enzyme solution, and T379S crude enzyme. Solution and M372I-T379S crude enzyme solution. The crude enzyme solution was separated and purified by affinity chromatography on a nickel column. The...

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Abstract

Belonging to the technical field of bioengineering, the invention relates to an aspartokinase mutant, an engineering bacterial strain and a preparation method of the mutant. The high enzyme activity aspartokinase mutant has an amino acid sequence formed by substitution, deletion or addition of 2-10, further 2-6, and even further 2-3 amino acid residues to an exogenous aspartokinase with an amino acid sequence shown as SEQ ID NO:2, and has the functions of aspartokinase. Preferably, the sequence is formed by substitution, deletion or addition of 2 amino acid residues. In particular, preferably,site-specific saturation mutation is carried out to the 372nd and 379th amino acids in the amino acid sequence, and a high throughput screening method is utilized to select the high enzyme activity aspartokinase mutant. The invention further utilizes the electrotransformation method to transfer a pEC-AK recombinant shuttle expression vector of the aspartokinase mutant that has high enzyme activity and can relieve or weaken Lys feedback inhibition effect into Corynebacterium pekinense, finally the recombinant Corynebacterium pekinense M372I-T379S engineering bacterial strain can be obtained, and fermentation product analysis is conducted on the engineering bacterial strain.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to aspartate kinase mutants, engineered bacteria and methods for preparing the mutants. Background technique [0002] Aspartokinase (AK) is widely present in bacteria, fungi and plants. It is a key rate-limiting enzyme in the aspartate amino acid synthesis pathway. It catalyzes the reaction between L-aspartic acid and adenosine triphosphate (ATP). L-aspartyl-4-yl phosphate and adenine nucleoside diphosphate (ADP) are produced. However, aspartate kinase is an allosteric enzyme, which is subject to the synergistic feedback inhibition of end products such as threonine and lysine, and can flexibly control the carbon flow according to the accumulation of the end product, thus limiting the aspartate family The accumulation of amino acids. [0003] Since aspartate kinase is strictly regulated by the end product, removing the feedback inhibition of the end product on AK is one ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/70C12N1/21
CPCC12N9/1217C12N15/70C12Y207/02004
Inventor 闵伟红高云娜方丽韩彩静
Owner JILIN AGRICULTURAL UNIV
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