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Recombinant corynebacterium glutamicum for producing gamma-polyglutamic acid as well as construction method and use of recombinant corynebacterium glutamicum

A technology of Corynebacterium glutamicum and polyglutamic acid, which is applied in the field of recombinant Corynebacterium glutamicum for producing γ-polyglutamic acid and its construction and use

Inactive Publication Date: 2013-06-12
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although great progress has been made in the fermentative production of γ-PGA, since the activity of the key enzyme (PgsB, γ-amide ligase) in the synthesis of γ-PGA needs to rely on glutamic acid, the fermentation production process needs time to time. Adding glutamic acid, so efficient and low-cost production of γ-PGA still needs further research

Method used

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  • Recombinant corynebacterium glutamicum for producing gamma-polyglutamic acid as well as construction method and use of recombinant corynebacterium glutamicum
  • Recombinant corynebacterium glutamicum for producing gamma-polyglutamic acid as well as construction method and use of recombinant corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Construction of recombinant Corynebacterium glutamicum C. glutamicum ATCC13869 (pEKEx2-BCA)

[0072] (1) Strains: Corynebacterium glutamicum C. glutamicum ATCC13869

[0073] (2) Foreign gene: plasmid pEKEx2-BCA

[0074] (3) Vector: E.coli / C.glutamicum shuttle vector pEKEx2 (Eggeling L, Bott M. Handbook of Corynebacterium glutamicum. CRC press, 2005, Boca Raton)

[0075] (4) Construction of recombinant plasmid pEKEx2-BCA

[0076] Using the genome of Bacillus subtilis TKPG011 as a PCR template, primers were designed to amplify pgsB, pgsC and pgsA, respectively. Connect both ends of the pgsB fragment to BamHI and HindⅢ sites respectively, the primers are: sense strand: 5'-gttgttggatccatgtggttatagcattatagcctgtg-3'; antisense strand: 5'-gttgttaagcttcatgcttacgagctgcttaacc-3', PCR amplification parameters are as follows: 95°C for 30s, 54°C for 50s, 72°C for 1min30s, a total of 30 cycles; connect the two ends of the pgsC fragment with HindⅢ and NdeI sites respecti...

Embodiment 2

[0079] Example 2: Fermentative production of γ-PGA by recombinant Corynebacterium glutamicum C. glutamicum ATCC13869 (pEKEx2-BCA)

[0080] (1) Fermentation strain: recombinant Corynebacterium glutamicum C. glutamicum ATCC13869 (pEKEx2-BCA)

[0081] (2) Two kinds of fermentation media

[0082] Biotin limited medium: 50g glucose, 30g (NH4) 2 SO 4 , 1g KH 2 PO 4 , 0.4g MgSO 4 ·7H 2 O, 0.01g FeSO4 7H 2 O, 0.01g MnSO 4 4H 2 O, 200μg vitamin B1HCl, 13.8ml soybean protein hydrolyzate (35g / L total nitrogen), 50gCaCO 3 (need to be sterilized separately), adjust the pH to 8.0 with KOH (the total volume is 1L).

[0083] Add Tween40 medium: 50g glucose, 30g (NH4) 2 SO 4 , 1g KH 2 PO 4 , 0.4g MgSO 4 ·7H 2 O, 0.01g FeSO4 7H 2 O, 0.01g MnSO 4 4H 2 O, 200μg vitamin B1HCl, 60μg biotin, 13.8ml soybean protein hydrolyzate (35g / L total nitrogen), 50g CaCO 3 (need to be sterilized separately), adjust the pH to 8.0 with KOH, and add Tween40 to a final concentration of 5mg / ml (th...

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Abstract

The invention discloses recombinant corynebacterium glutamicum for producing gamma-polyglutamic acid as well as a construction method and a use of the recombinant corynebacterium glutamicum. The recombinant corynebacterium glutamicum is characterized by being an engineering bacteria which is obtained by transferring a recombinant expression plasmid containing a gamma-PGA (Poly Glutamic Acid) synthetase composite gene pgsBCA to an original strain wild C.glutamicum ATCC13869. The recombinant corynebacterium glutamicum disclosed by the invention contains the gamma-PGA synthetase composite gene pgsBCA, so that much gamma-PGA can be synthesized under biotin limit or under the fermentation condition of adding Tween40, therefore, the glutamic acid adding cost in the gamma-PGA production process is reduced, and a foundation is laid for the innovation in realizing the one-step production of gamma-PGA better.

Description

technical field [0001] The invention relates to a recombinant corynebacterium glutamicum producing gamma-polyglutamic acid, its construction method and application. Background technique [0002] Corynebacterium glutamicum was originally isolated by Kinoshita and colleagues in the soil environment. It is in the shape of short rods to small rods, sometimes slightly curved, with blunt ends, unbranched, single or in a figure-eight arrangement, and the bacteria 0.7~0.9×1.0~2.5 microns, Gram-positive, no spores, no movement, moist and round colonies, widely present in soil, ditch water, compost and around food factories, breweries, soy sauce factories, and monosodium glutamate factories. C. glutamicum utilizes glucose, fructose, mannose and sucrose as carbon and energy sources for growth and grain production through the phosphoenolpyruvate sugar transport system (PTS system) under biotin-limited, Tween40 or penicillin-added conditions Fructose (Dominguez H, Lindley ND. Complete s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/02C12R1/15C12R1/125
Inventor 姚文娟孟国梁张伟陈向凡殷润婷
Owner NANTONG UNIVERSITY
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