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High enzyme activity aspartokinase mutant, engineering bacteria and preparation method of the mutant

A technology of aspartokinase and mutants, which is applied in the field of bioengineering and can solve problems such as heavy tasks, long cycles, and difficulty in further increasing the yield of target substances.

Active Publication Date: 2021-12-17
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 through random mutation and subsequent screening through the action of physical or chemical mutagens, such as screening resistance to structural analogs and auxotrophic mutant strains, but the above methods have heavy tasks, long cycles, and it is difficult to further improve the target production volume etc.

Method used

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  • High enzyme activity aspartokinase mutant, engineering bacteria and preparation method of the mutant
  • High enzyme activity aspartokinase mutant, engineering bacteria and preparation method of the mutant
  • High enzyme activity aspartokinase mutant, engineering bacteria and preparation method of the mutant

Examples

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Effect test

Embodiment 1

[0035] Embodiment 1, the construction of recombinant escherichia coli strain

[0036] (1) Cloning of AK gene

[0037] Using the TAKaRa Genome Extraction Kit to extract the chromosomal DNA of Corynebacterium pekinense, use it as a template to carry out PCR amplification under the action of cloning primers, clone a large number of AK target gene fragments, and verify by PCR nucleic acid electrophoresis, Corynebacterium pekinense Acid kinase (AK) in the figure 1 There are bright bands between 1000 and 2000 bp in the middle, which is consistent with the length of SEQ ID NO:1. All reagents were purchased from TAKaRa Company, and the designed cloning primers were 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 cutting site of NdeI and EcoRI restriction enzymes.

[0041]

[0042] (2) Construction of recombinant Escherichia co...

Embodiment 2

[0059] Embodiment 2, construction of aspartokinase mutant

[0060] (1) Construction of aspartokinase M372 site mutant

[0061] Using the pET-AK recombinant plasmid in Example 1 (2) as a template, primer 5'-GTAACACCTGGGTGAGACTG upstream of M372N NNN GCCCGCACC-3', downstream primer 5'-CTCGT GGGTGCGGGC NNN Under the action of CAGTCTCACCC-3' (the underlined part is the mutation site), the mutation PCR reaction is carried out, and the PCR product and the pET-28a vector are double-digested by restriction endonucleases EcoRI and NdeI and then ligated overnight (using Example 1 (2) in the double enzyme digestion and ligation system), the ligated product was transferred into Escherichia coli BL21 (DE3) competent by implementing the method of 1 (2), and aspartokinase M372 site mutant was obtained strain.

[0062]

[0063]

[0064] (2) Construction of aspartokinase T379 site mutant

[0065] Using the pET-AK recombinant plasmid in Example 1 (2) as a template, using the PCR react...

Embodiment 3

[0074] Embodiment 3, enzyme kinetic analysis and enzymatic property characterization

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

[0076] The recombinant escherichia coli strain (WT) of embodiment 1 (2) was inoculated with the high enzyme activity mutant (single mutant M372I and T379S) and double mutant M372I-T379S in embodiment 2 with 2% (v / v) The amount was transferred to 100mL LB liquid medium containing kanamycin, cultured overnight at 37°C, 180r / min, then added IPTG inducer (final concentration 1mmol / L), induced at 30°C, 130r / min for 12h . The bacterial solution was centrifuged at 8000r / min for 10min, the supernatant was discarded, and 10mL of PBS was added to resuspend the bacteria, and the supernatants obtained by ultrasonication for 30min and centrifugation at 8000r / min for 10min were respectively WT crude enzyme solution, M372I crude enzyme solution, and T379S crude enzyme solution. solution and M372I-T379S crude enzyme solution. The crude enzym...

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Abstract

An aspartokinase mutant, an engineering bacterium and a preparation method of the mutant belong to the technical field of bioengineering. The high-enzyme activity aspartokinase mutant of the present invention is an exogenous aspartokinase whose amino acid sequence is shown in SEQ ID NO: 2 after 2-10, further 2-6, and further 2 A sequence formed by substitution, deletion or addition of ~3 amino acid residues, and has the function of aspartokinase. A sequence formed by substitution, deletion or addition of two amino acid residues is preferred, and it is particularly preferred to perform site-directed saturation mutations on amino acids at positions 372 and 379 in the amino acid sequence, and use a high-throughput screening method Selection of high enzymatic activity aspartokinase mutants. The present invention further utilizes the electroporation method to transfer the pEC-AK recombinant shuttle expression vector of the aspartokinase mutant with high enzyme activity and release or weaken Lys feedback inhibition into Corynebacterium Beijing, and finally obtain the recombinant Corynebacterium Beijing M372I ‑T379S engineering bacteria, and its fermentation product analysis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an aspartokinase mutant, an engineering bacterium and a preparation method of the mutant. Background technique [0002] Aspartokinase (Aspartokinase, AK) widely exists in bacteria, fungi and plants, and is the key rate-limiting enzyme in the aspartate family amino acid synthesis pathway, which catalyzes the reaction between L-aspartic acid and adenosine triphosphate (ATP). Generates L-aspartyl-4-yl phosphate and adenosine diphosphate (ADP). However, aspartokinase is an allosteric enzyme that is subject to cooperative feedback inhibition by terminal products such as threonine and lysine, which can flexibly control carbon flux according to the accumulation of end products, thus limiting the aspartate family. accumulation of amino acids. [0003] Since aspartokinase is strictly regulated by end products, releasing the feedback inhibition of AK by end products is...

Claims

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Application Information

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Patent Type & Authority Patents(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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