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Recombinant bacterial strain for high-yield production of L-lysine, and construction method and application of recombinant bacterial strain

A technology of recombinant strain and construction method, which is applied in the fields of genetic engineering and microorganisms, can solve the problems of hindering the utilization of carbohydrates by bacterial cells, affecting the accumulation of L-lysine in the bacterial cells, etc., so as to reduce production costs and achieve good bacterial stability. Effect

Active Publication Date: 2019-12-24
NINGXIA EPPEN BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, excessive NADPH in the cell will hinder the utilization of sugar by the cell, affecting the growth of the cell and the accumulation of L-lysine

Method used

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  • Recombinant bacterial strain for high-yield production of L-lysine, and construction method and application of recombinant bacterial strain
  • Recombinant bacterial strain for high-yield production of L-lysine, and construction method and application of recombinant bacterial strain
  • Recombinant bacterial strain for high-yield production of L-lysine, and construction method and application of recombinant bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Constructing the transformation vector pK18-pobB (G139A, G811A) of the coding region of the pobB gene comprising a point mutation

[0052] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, three pairs of primers for amplifying the coding region of pobB gene were synthesized, and allelic replacement was performed in strain YP097158 (preservation number: CGMCC No.12856, date of preservation: August 2016) On March 16, depository unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.) The introduction point in the pobB gene coding region (SEQ ID No.1) in the background Mutation, the 139th G of the nucleotide sequence of the pobB gene is changed to A, the 811th G is changed to A (SEQ ID No.2), and the amino acid sequence of the corresponding encoded protein is changed from wild type (SEQ ID NO.3) to 47th Glycine (G) at position 271 i...

Embodiment 2

[0062] Example 2 Construction of pobB comprising point mutations (G139A,G811A) engineered strain

[0063] Construction method: allelic replacement vector pK18-pobB (G139A,G811A) Transformed into the patented strain YP097158 of L-lysine producing bacteria by electric shock (for its construction method, please refer to the construction method of engineering strains in WO2014121669A1; it was confirmed by sequencing that the wild-type pobB gene coding region was retained on the chromosome of the strain), and the production of The single colonies were identified by primer P1 and universal primer M13F respectively, and the strains that could amplify bands with a size of about 1600 bp were positive strains. The positive strains were cultured on a medium containing 15% sucrose, and the single colony produced by the culture was cultured on a medium containing kanamycin and a medium not containing kanamycin, respectively, and on a medium not containing kanamycin The strains that do no...

Embodiment 3

[0069] Example 3 L-lysine fermentation experiment

[0070] The bacterial strain YPL-4-012 constructed in Example 2 and the original bacterial strain YP097158 were used in the BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium shown in Table 1 and Table 2 Fermentation experiments were carried out with the control process shown. Each strain was repeated three times, and the results are shown in Table 3.

[0071] Table 1 Fermentation Medium Formula

[0072] Element formula starch hydrolyzed sugar 30g / L ammonium sulfate 12g / L magnesium sulfate 0.87g / L molasses 20g / L acidified corn steep liquor 3mL / L phosphoric acid 0.4mL / L potassium chloride 0.53g / L Defoamer (2% foam enemy) 4mL / L ferrous sulfate 120mg / L Manganese sulfate 120mg / L Nicotinamide 42mg / L thbrthdrexvbdr 6.3mg / L Vitamin B1 6.3mg / L Copper, zinc salt solution 0.6g...

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Abstract

The invention provides a recombinant bacterial strain for high-yield production of L-lysine, and a construction method and application of the recombinant bacterial strain. A polynucleotide sequence comprises a modified pobB gene coding polynucleotide sequence as shown in SEQ ID NO:2, and the invention provides a recombinant protein coded by the polynucleotide sequence, the recombinant bacterial strain containing the polynucleotide sequence, a construction method of the recombinant bacterial strain, and an application of the recombinant bacterial strain in fermentation production of L-lysine. According to the recombinant strain disclosed by the invention, point mutation is carried out on the nucleotide sequence of an open reading frame coding region of the pobB gene in wild-type corynebacterium glutamicum to obtain the recombinant strain modified by the coding region of the pobB gene, and the yield of L-lysine is realized compared with the yield of L-lysine by using wild bacterial strains.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and microbes, and in particular relates to a high-yield L-lysine recombinant strain and its construction method and application. Background technique [0002] L-Lysine is one of the eight amino acids necessary for human and animal life activities. L-lysine has a variety of physiological functions, such as regulating metabolic balance, promoting human development, promoting the body's absorption of cereal protein and other amino acids, enhancing immune function, etc., and is widely used in food additives, animal feed, drug synthesis, etc. aspect. [0003] The production methods of L-lysine mainly include proteolysis and microbial fermentation. Among them, the microbial fermentation method has been called the most commonly used method for industrially producing L-lysine because of the advantages of easy control of the production process and high production capacity. The microbial fer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/74C12N1/21C12P13/08C12R1/15C12R1/19
CPCC07K14/34C12N15/70C12N15/74C12P13/08
Inventor 孟刚马风勇周晓群贾慧萍苏厚波杨立鹏高晓航田斌
Owner NINGXIA EPPEN BIOTECH
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