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Method for improving yield of arginine of corynebacterium crenatum by synthesis way through knocking out proline

A technology of Corynebacterium blunt tooth, proline, applied in the field of biology, can solve the problem that the genetic background is in a blank state

Inactive Publication Date: 2013-06-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Corynebacterium crenatum is a cognate, non-spore-forming, Gram-positive bacterium isolated by researchers in my country. Its mutant strains are widely used in domestic amino acid production, but the study of its genetic blank

Method used

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  • Method for improving yield of arginine of corynebacterium crenatum by synthesis way through knocking out proline
  • Method for improving yield of arginine of corynebacterium crenatum by synthesis way through knocking out proline
  • Method for improving yield of arginine of corynebacterium crenatum by synthesis way through knocking out proline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Detection of glutamic acid kinase enzyme activity in the recombinant corynebacterium bacillus

[0024] The proB gene-deleted transformants verified above were randomly selected and cultured in the seed medium to the logarithmic growth phase, and the bacterial cells were collected and ultrasonically disrupted to measure the activity of intracellular glutamate kinase: with L-glutamic acid as the substrate, Add 50mM L-glutamic acid, 10mM ATP, 20mM MgCl in 0.25mL system with pH 7.0 2 , 100mM hydroxylammonium hydrochloride, 50mM Tris, add 200μL of crude enzyme solution, mix thoroughly, place at 37°C, and react for 30min. FeCl with trichloroacetic acid added 3 1 mL of the reaction termination solution was centrifuged at 3,500 g at a low speed, and the supernatant was taken to measure the absorbance of γ-glutamic acid hydroxamic acid at 535 nm. 1 U is defined as the amount of enzyme required to generate 1 μmol of hydroxamate per minute. Compared with the contr...

Embodiment 2

[0025] Example 2: Shake flask fermentation of recombinant Corynebacterium blunt-toothed ΔproB transformants to produce L-arginine

[0026] Seed medium (g / L): glucose 30, corn steep liquor 20, urea 1.5, KH 2 PO 4 1, (NH 4 ) 2 SO 4 20, MgSO 4 ·7H 2 O 0.5; pH 7.0-7.2, filling volume 30mL / 250mL, sterilize at 121°C for 20min.

[0027] Fermentation medium (g / L): glucose 150 (divided), corn steep liquor 40, biotin 8×10 -5 , L-histidine 5×10 -4 , MnSO 4 ·H 2 O 0.02, (NH 4 ) 2 SO 4 20, MgSO 4 ·7H 2 O 0.5, KH 2 PO 4 1.5, FeSO 4 ·7H 2 O 0.02, CaCO 3 30 (minus sterilization). The pH is 7.0-7.2, the filling volume is 25mL / 250mL, and it is sterilized at 121°C for 10 minutes.

[0028] The above-mentioned recombinant bacteria Corynebacterium blunt toothed SYPA 5-5ΔproB, which has been verified by enzyme activity, was subjected to a shake flask fermentation experiment, and a ring of Coryne bacillus blunt toothed bacteria (control bacteria and recombinant bacteria) was pick...

Embodiment 3

[0029] Example 3: Recombinant Corynebacterium blunt-toothed ΔproB transformant fermented in a 5L fermenter to produce acid

[0030] Seed medium (g / L): glucose 30, corn steep liquor 20, urea 1.5, KH 2 PO 4 1, (NH 4 ) 2 SO4 20, MgSO 4 ·7H 2 O 0.5; pH 7.0-7.2, filling volume 30mL / 250mL, sterilize at 121°C for 20min.

[0031] Fermentation medium (g / L): glucose 150 (divided), corn steep liquor 40, biotin 8×10 -5 , L-histidine 5×10 -4 , MnSO 4 ·H 2 O 0.02, (NH 4 ) 2 SO 4 20, MgSO 4 ·7H 2 O 0.5, KH 2 PO 4 1.5, FeSO 4 ·7H 2 O 0.02, CaCO 3 30 (minus sterilization). pH is 7.0~7.2, liquid volume is 25mL / 250mL, sterilized at 121℃ for 10min [93] .

[0032] Batch fermentation conditions in 5L self-controlled fermenter: liquid volume in 5L fermenter is 3L, inoculum size is 5%, 30℃, ventilation rate is 3L / min, ammonia water is added automatically to control pH at 6.8, factory fermentation of L-fine Special antifoaming agent for amino acid, automatic control of dissolved ...

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Abstract

L-arginine is a semi-essential basic amino acid in human bodies and animal bodies, and has multiple unique physiological and pharmacological effects. The invention mainly aims at knocking out a competitive bypass metabolism key enzyme gene for proline synthesis, wherein a corynebacterium crenatum genome serving as a template is subjected to PCR (polymerase chain reaction) amplification respectively by using upstream and downstream primers of a proB gene, and is used for preparing a deletion type gene proB' by utilizing overlap PCR amplification; recombinant plasmid pK18mobsacB-proB' is connected and electroporated to enter the corynebacterium crenatum, and a secondary homologous recombination gene deletion type strain is screened; the recombinant strain corynebacterium crenatum delta proB is subjected to a flask shaking experiment, and functions of the proB gene in the corynebacterium crenatum are lost, so that the capability of proline synthesis is greatly reduced, and sugar consumption can be improved; and the accumulation of proline in the fermentation broth is only 2.7 percent of that of a control strain, so that the yield of target amino acid L-arginine can be improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing arginine by industrial microorganisms. More specifically, the present invention relates to a method for producing arginine by genetically engineered recombinant Corynebacterium cretinus. Background technique [0002] L-Arginine is a semi-essential basic amino acid in humans and animals. It is an important raw material for the synthesis of protein creatine and an important intermediate metabolite of the urea cycle in organisms. It has a variety of unique physiological and pharmacological effects. L-arginine is widely used in clinical medicine, food, cosmetics and related biological research fields. Fermentation is currently a relatively effective and economical method for the commercial production of L-arginine. [0003] With the improvement of the nutritional level of medicine in our country, the demand for arginine is increasing day by day. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/10C12N1/21C12P13/10C12R1/15
Inventor 饶志明许正宏徐美娟孙红梅窦文芳
Owner JIANGNAN UNIV
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