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Method for increasing yield of L-arginine synthesized from corynebacterium crenatum

A technology of Corynebacterium blunt and arginine, which is applied in the field of bioengineering to achieve the effect of optimizing fermentation conditions

Active Publication Date: 2019-02-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 studies on its genetic background still blank

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Knockout of Nitrogen Transcription Regulator AmtR

[0026] Using the genomic DNA of Corynebacterium bacillus SYPA5-5 as a template, amtR F1 (sequence shown in SEQ ID NO.3) and R1 (sequence shown in SEQ ID NO.4), amtR F2 (sequence shown in SEQ ID NO. .5) and R2 (sequence shown in SEQ ID NO.6) were used as primers to amplify the upstream and downstream two homologous arms of about 500bp each, and then perform fusion extension PCR to obtain a gene fragment with a size of 1200bp, which was connected to the pK18mobsacB vector The pk18-amtR was obtained, the pk18-amtR was sequenced, and the sequence was compared by DNAMAN, and the result showed that the gene sequence was correct. The recombinant plasmid pk18-amtR was electrotransferred into Corynebacterium blunt-toothed SYPA5-5, and the grown single colony was expanded and cultured, transferred to a plate containing sucrose for screening, and colony PCR was used for verification, and the amtR gene was completely kn...

Embodiment 2

[0027] Example 2: Cloning and expression of ammonium transporter AmtB in Corynebacterium crimsonum strain Cc5-5

[0028] The genomic DNA of Corynebacterium bacilli SYPA5-5 was used as a template and the primer amtBF1 / R1 was used for PCR amplification to obtain a gene fragment of 1317bp. After double digestion with HindIII and EcoRI, the amtB fragment was recovered, ligated with pXMJ19 linearized with the same enzyme, and then transformed into Escherichia coli BL21, the positive transformant was picked to extract the plasmid, and the plasmid was used as a template for PCR verification. The results showed that the plasmid pXMJ19-amtB was constructed successfully. The recombinant plasmid pXMJ19-amtB was electrotransformed into Corynebacterium bacillus Cc5-5 to obtain the recombinant strain Cc5-5 / pXMJ19-amtB.

[0029] Cultivate the recombinant strain Cc5-5 / pXMJ19-amtB in a 50mL container containing 10mL LBG medium for 16-20h, and then transfer it to a 250mL container containing 5...

Embodiment 3

[0030] Embodiment 3: the fermentation of recombinant bacteria Cc5-5 / pXMJ19-amtB

[0031] (1) Seed cultivation

[0032]Pick a single colony of recombinant Corynebacterium bacilli Cc5-5 / pXMJ19-amtB from the activation plate and inoculate it in 10mL LBG medium (containing 100mg / ml chloramphenicol), culture it with shaking at 30°C for 24h, and then transfer it to 150ml In the seed medium, culture at 30°C for 24h.

[0033] (2) Fermentation culture

[0034] The initial fermentation culture volume is 2.5L, and the components of the fermentation medium used are as follows:

[0035] Fermentation medium components: glucose 150g / L, ammonium sulfate 40g / L, yeast extract 15g / L, magnesium sulfate heptahydrate 0.5g / L, potassium chloride 1g / L, potassium dihydrogen phosphate 1.5g / L, heptahydrate Ferrous sulfate 0.02g / L, manganese sulfate monohydrate 0.02g / L, prepared with deionized water, pH 7.0. The pH of the above fermentation medium was adjusted to 7.0 with 50% ammonia water, and steril...

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Abstract

The invention discloses a method for increasing the yield of L-arginine synthesized from corynebacterium crenatum, and belongs to the technical field of bioengineering. By means of the method, recombinant corynebacterium crenatum of which a nitrogen transcriptional regulation factor AmtR is knocked out and ammonium transport protein is over-expressed is successfully constructed. By adopting a strategy of batch fermentation by a 5-L fermentation tank and optimizing fermentation conditions, finally the recombinant corynebacterium crenatum Cc5-5 / pXMJ19-amtB is fermented for 96 h, the L-arginine yield of the recombinant corynebacterium crenatum reaches 60.9+ / -1.31 g / L, the yield is increased by 30.56% compared with an original strain of corynebacterium crenatum original strain SYPA5-5, the production intensity reaches 0.634 g / L.h, and the saccharic acid conversion rate is 0.36+ / -0.018 g / g, so that high yield of the L-arginine is realized. Meanwhile, utilization of NH4+ is increased in therecombinant corynebacterium crenatum, so that it is shown that knockout of the nitrogen transcriptional regulation factor and overexpression of the ammonium transport protein have a remarkable effecton the yield of the L-arginine.

Description

technical field [0001] The invention relates to a method for improving the production of L-arginine synthesized by Coryne bacilli, especially a method for improving the production of L-arginine synthesized by Coryne bacillus by overexpressing the ammonium transporter AmtB, which belongs to bioengineering technology field. Background technique [0002] N element in biological cells is the second largest macro element and an important component of proteins and nucleotides. The synthesis of substances in cells is inseparable from the uptake and utilization of N sources. Nitrogen accounts for 32.1% in L-arginine, which is the amino acid with the highest N:C ratio among natural amino acids, and can be used as an important nitrogen source supply in biological cells. L-arginine is an important raw material for the synthesis of protein and creatine, a semi-essential amino acid for humans and animals, and an essential amino acid for the growth and development of infants and young c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/10C12N15/77C12R1/15
CPCC07K14/34C12N15/77C12P13/10
Inventor 徐美娟饶志明李静舒群峰张显杨套伟
Owner JIANGNAN UNIV
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