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Construction method and application of over-expressing fusA genetic engineering bacterium

A technology of genetically engineered bacteria and overexpression, applied in the fields of genetic engineering and microbial fermentation, can solve problems such as poor safety, low yield, and high cost, and achieve the effects of reducing the difficulty of separation and purification, reducing production costs, and increasing production

Active Publication Date: 2014-12-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amino acid components of natural proteins are complex and difficult to extract and separate, so this method is rarely used in production at present
Although the chemical synthesis method has high purity, the reaction requires high temperature and high pressure conditions, high energy consumption, high cost, low yield, and the use of toxic and harmful organic solvents is involved in the production process, which has poor safety. To obtain nutritional value Amino acids must be resolved into optical isomers
However, this splitting process is complicated and the yield is low, so it is generally not used.

Method used

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  • Construction method and application of over-expressing fusA genetic engineering bacterium
  • Construction method and application of over-expressing fusA genetic engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of Elongation Factor EF-G Genetic Engineering Bacteria

[0026] Proceed as follows:

[0027] (1) Design primers for amplification FusA

[0028] Upstream primer: 5'-CTA GCTAGC A GAAGGAGTAA ATCGGTGGCT CAAGAAGTGC TTAAG-3', underlined Nhe I restriction site;

[0029] Downstream primer: 5'-AACC CTCGAG TTAGGAAGCG GTGCCGTTGC-3', underlined as xho I restriction site;

[0030] (2) Use the PCR product and the expression vector pDXW-8 with Nhe I / xho I double digestion, after recovery, the FusA and pDXW-8 at a molar ratio of 6:1 to 3:1, and were ligated with T4 ligase at 16°C for 2 hours to construct the recombinant expression vector pDXW-8- FusA (Such as figure 1 );

[0031] (3) Then the recombinant plasmid pDXW-8- FusA Introduced into Corynebacterium glutamicum WY001, cultured in LBHIS solid medium containing kanamycin (50 μg / mL) for 48 hours at 30° C., and screened for transformants;

[0032] (4) Extract the plasmids of the screened t...

Embodiment 2

[0033] Example 2 Overexpression of EF-G Genetic Engineering Bacteria in Shake Flasks to Produce Amino Acids

[0034] From the deposited strain WY001 / pDXW-8- FusAStretch a ring of bacterial solution in a glycerol tube and streak it on the solid activation medium, and incubate at 30°C for 36 h. Use an inoculation loop to scrape an inoculation loop lawn from the activated plate and transfer it to a 250 mL Erlenmeyer flask containing 25 mL of seed medium, and incubate at 30 °C and 200 rpm for 18 h. Take 2.4 mL of bacterial liquid from the cultured seed liquid and transfer it to a 250 mL Erlenmeyer flask containing 23 mL of fermentation medium. After culturing for 6 h at 30 °C, add a final concentration of 1 mM isopropylthiogalactoside ( IPTG) induced protein expression until the end of fermentation (t=72h).

[0035] After the fermentation, the fermented liquid adopts HPLC to measure amino acid content, and utilizes HPLC OPA (o-phthalaldehyde) pre-column derivatization me...

Embodiment 3

[0042] Example 3 Overexpression of EF-G Genetic Engineering Bacteria to Produce Amino Acids in a Fermenter

[0043] From the deposited strain WY001 / pDXW-8- FusA Stretch a ring of bacterial solution in a glycerol tube and streak it on the solid activation medium, and incubate at 30°C for 36 h. Use an inoculation loop to scrape an inoculation loop bacterial lawn from the activated plate and put it into a 500 mL Erlenmeyer flask containing 50 mL of seed medium, and incubate at 30 °C and 200 rpm for 18 h. Transfer 150 mL of seed solution to a 3 L fully automatic fermenter with 1.2 L of fermentation medium. Use 50% ammonia water to automatically adjust pH=7.0, use cooling circulating water and base heating plate to automatically control temperature to 30°C, use stirring speed and dissolved oxygen coupling to maintain dissolved oxygen at 30%, and ventilation rate (1.5 vvm). When cultured for 6 h, protein expression was induced by adding a final concentration of 1 mM IPTG. ...

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Abstract

The invention relates to a construction method and an application of an over-expressing fusA genetic engineering bacterium, and belongs to the technical fieldof gene engineering and microbial fermentation. The method comprises the following steps: cloning elongation factor EF-G gene fusA through utilizing PCR amplification, connecting a gene fragment to an over-expression vector pDXW-8, electrically transferring to Corynebacterium glutamicum WY001, and carrying out antibiotic resistance screening to obtain the target engineering bacterium WY001 / pDXW-8-fusA. The Corynebacterium glutamicum gene engineering bacterium constructed in the invention can improve the output of amino acids in shake flask fermentation or fermentation cylinder fermentation, and is in favor of reducing the separation and purification difficulty and improving the yield in order to substantially reduce the production cost of the amino acids.

Description

technical field [0001] The present invention relates to a strain overexpressing FusA The invention relates to a method for constructing genetically engineered bacteria and its application for increasing the yield of amino acids, belonging to the technical field of genetic engineering and microbial fermentation. Background technique [0002] Amino acid is one of the many active macromolecules that build organisms, and is the basic material for building cells and repairing tissues. Amino acids provide energy for body and brain activity. There are 21 kinds of basic amino acids that make up protein. There are 8 kinds of amino acids that cannot be synthesized by the human body and must be obtained from food. They are essential amino acids for the human body; Get it from food. The balance and proper supply of amino acids are the basic premise of human health. The lack of any amino acid will affect the immune system and other normal functions. Therefore, amino acids are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12P13/08C12P13/06C12R1/15C12N1/21
Inventor 王小元赵建勋胡晓清李颜颜尹良鸿
Owner JIANGNAN UNIV
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