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Method for improving yield of L-arginine synthesized by corynebacterium crenatum

A technology of Coryne bacilli and amino acids, applied in the field of bioengineering

Inactive Publication Date: 2019-06-28
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

[0031] Example 1: Transformation of bifunctional uridine acyl transfer / removal enzyme GlnD by integration mutation

[0032] Using the genomic DNA of Corynebacterium bacillus SYPA5-5 as a template, with F1 (sequence shown in SEQ ID NO.5) and Rm (sequence shown in SEQ ID NO.6), Fm (sequence shown in SEQ ID NO.7 Shown) and R1 (sequence shown in SEQ ID NO.8) were used as primers to amplify the upstream and downstream two homology arms by PCR, and fusion extension PCR was performed to obtain a 2137bp gene fragment, which was connected to the pK18mobsacB vector to obtain pk18-glnD AA , the pk18-glnD AA Sequencing and sequence comparison by DNAMAN showed that the mutant gene sequence was correct. The recombinant plasmid pk18-glnD AA Electrotransfer into Corynebacterium blunt tooth SYPA5-5, expand the grown single colony, transfer to a plate containing sucrose for screening, use colony PCR for verification, and obtain glnD after screening AA The mutant strain successfully integrate...

Embodiment 2

[0033] Example 2: pII signal transduction protein GlnK in Corynebacterium bacillus Cc-HD AA and cloned expression in Corynebacterium bacilli SYPA5-5

[0034] Using the genomic DNA of Corynebacterium bacillus SYPA5-5 as a template, the primer glnKF1 / R1 was used for PCR amplification to obtain a 339bp gene fragment. After double digestion with HindIII and EcoRI, the glnK 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-glnK was constructed successfully. Electrotransformation of the recombinant plasmid pXMJ19-glnK into Corynebacterium bacilli toothonus strain Cc-HD AA and SYPA5-5, the recombinant strain Cc-HD was obtained AA / pXMJ19-glnK and Cc / pXMJ19-glnK.

[0035] Cultivate the two strains of recombinant bacteria in a 50mL container con...

Embodiment 3

[0036] Embodiment 3: Recombinant bacteria Cc-HD AA / Fermentation of pXMJ19-glnK

[0037] (1) Seed cultivation

[0038] Recombinant Corynebacterium blunt-toothed Cc-HD was picked from the activated plate AA A single colony of / pXMJ19-glnK was inoculated in 10 mL of LBG medium (containing 100 mg / mL chloramphenicol), cultured with shaking at 30°C for 24 hours, and then all were transferred into 150 mL of seed medium, and cultured at 30°C for 24 hours.

[0039] (2) Fermentation culture

[0040] The initial fermentation culture volume is 2.5L, and the components of the fermentation medium used are as follows:

[0041] 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 ...

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Abstract

The invention discloses a method for improving the yield of L-arginine synthesized by corynebacterium crenatum, specifically discloses a method for improving the yield of L-arginine through integratedmutation modification of bi-functional uridyl transfer / removal enzyme GlnD, modification of pII signal transduction protein GlnK and uridine acylation of GlnK, and belongs to the technical field of bioengineering. The method successfully achieves the integrative mutation modification of the bifunctional uridyl transfer / removal enzyme GlnD, the pII signal transduction protein GlnK is overexpressedon this base, and the uridine acylation of GlnK is enhanced. A 5-L fermentation tank is used for batch fermentation, the fermentation conditions are optimized, finally the final recombinant corynebacterium crenatum Cc-HDAA / pXMJ19-glnK is fermented for 96 h, the yield of L-arginine of the recombinant bacteria reaches 52.2 g / L, the production intensity reaches 0.544 g / L h, and the high yield of L-arginine is achieved.

Description

technical field [0001] The present invention relates to a method for increasing the production of L-arginine synthesized by Corynebacterium blunt tooth, in particular to a method for transforming bifunctional uridylyl transfer / removal enzyme GlnD through integration mutation, and modifying pII signal transduction protein GlnK uridylylation The invention relates to a method for increasing the output of L-arginine, which belongs to the technical field of bioengineering. Background technique [0002] Nitrogen is the material that constitutes macromolecular nucleic acid, protein and other nitrogen compounds in organisms, and is one of the most basic growth factors for various microorganisms. After long-term evolution, microorganisms have formed a very fine global nitrogen regulation network to cope with the survival pressure in different environments. NH 4 + It is the basic component of the medium and the most preferred nitrogen source for most microorganisms. [0003] Coryn...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N1/21C12P13/10C12R1/15
Inventor 徐美娟饶志明李静舒群峰张显杨套伟
Owner JIANGNAN UNIV
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