Engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and construction method and application of engineering bacterium

A technology of engineering bacteria and pyruvate, applied in the field of metabolic engineering, can solve problems such as long catalysis time

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the strain has a long catalytic time, and

Method used

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  • Engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and construction method and application of engineering bacterium
  • Engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and construction method and application of engineering bacterium
  • Engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and construction method and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Molecular docking and selection of saturation mutation sites

[0038] Search the crystal model of its homologous enzyme in the SWISS MODEL database. Model 5fjm.1A has high homology with pm1, and its homology is 93.51%. Comparing its amino acid series with that of pm1, the results show that only a few regions have different amino acids, which shows that the crystal structure of 5fjm.1A is basically consistent with that of pm1. Therefore, model 5fjm.1A was selected for molecular docking with L-alanine.

[0039] Semi-flexible molecular docking experiments were performed using Autodock software. According to the operation manual of the Autodock software, the three-dimensional protein structure file of the model 5fjm.1A (including the structure of the coenzyme FAD) and the three-dimensional structure file of the ligand L-alanine were molecularly docked using the Lamarck genetic algorithm (LGA).

[0040] Select the characteristic sites of site-directed saturation ...

Embodiment 2

[0041] Example 2: Establishment of Site-Directed Saturation Mutation Library

[0042] Table 1

[0043]

[0044] According to the gene sequence of pm1, design the primers shown in the above table, use the above primers and use the pET20b-pm1 plasmid as a template to amplify the whole plasmid by PCR, transfer the PCR products into E. on the tablet.

Embodiment 3

[0045] Example 3: High-throughput screening and validation

[0046] Use a toothpick to pick about 100 single clones from each saturated mutation point library and transfer them to a 96-well plate for seed culture. Each well contained 600 μl of ampicillin-resistant liquid LB medium, and cultured at 37° C. for 12 hours on a well plate shaker. Afterwards, 200 μl of the seed solution was transferred to a 96-well plate containing 600 μl of ampicillin-resistant liquid TB medium per well for fermentation induction culture, and at the same time, IPTG (final concentration 0.01 mM) was added to induce at 37° C. for 6 h and then the fermentation was terminated. Collect the cells by centrifugation (3,500rpm, 5min, 4°C) in the fermentation broth, discard the supernatant and add 200μl 40g / L L-alanine (dissolved in 0.2M pH 7.0 phosphate buffer), and place on a plate shaker at 37°C The reaction was started in 30 min, and the reaction was stopped by centrifugation after 30 min, and the conten...

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PUM

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Abstract

The invention discloses an engineering bacterium for efficiently synthesizing pyruvic acid and D-alanine and a construction method and application of the engineering bacterium. On the basis of a genetic engineering strain BLK07 (pET20b-pm1), a strain with improved enzyme activity of pm1 is screened out through site-saturation mutagenesis, and an N-terminal encoding sequence (NCS) is inserted intoan N terminal of a gene sequence of the pm1, so that the protein expression quantity of the pm1 is improved, the yield of the pyruvic acid is increased, the catalytic time is significantly shortened and the catalytic efficiency is improved. When the concentration of substrates D-alanine and L-alanine is 110 g/L, the cell concentration is 4 g/L and a catalyst system only carries out catalysis at 37DEG C and 220 rpm in a shake flask for 8 h, the yield of the pyruvic acid can reach a maximum value 46.5 g/L, the conversion rate of the L-alanine is 84.5% and the resolution rate of the D/L-alaninereaches 84.5%.

Description

technical field [0001] The invention relates to an engineering bacterium for efficiently synthesizing pyruvate and D-alanine, a construction method and application thereof, and belongs to the technical field of metabolic engineering. Background technique [0002] Pyruvic acid (Pyruvic acid) is an important intermediate in the field of drug synthesis, widely used in food, pesticide, biochemical and other industries. At present, the industrial production methods of pyruvate mainly include chemical synthesis, enzymatic conversion and microbial fermentation. Among them, the cost of the chemical synthesis method is relatively high, and its pollution to the environment is relatively large; while in the microbial direct fermentation method, the components of the product are complex and the separation is difficult. Biocatalytic synthesis of pyruvate can overcome the shortcomings and shortcomings of the above two methods, so it has broad prospects in industrial applications. [000...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/53C12P41/00C12P7/40C12P13/06C12R1/19
CPCC12N9/0022C12N15/70C12P41/001C12P41/002C12P7/40C12P13/06C12Y104/03002
Inventor 刘龙陈坚堵国成李江华吕雪芹刘克周璇曲丽莎
Owner JIANGNAN UNIV
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