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Recombinant corynebacterium glutamicum and application thereof in producing L-glutamic acid

A technology of Corynebacterium glutamicum and glutamic acid, applied in the biological field, can solve the problems of restricting the industrial production process of L-glutamic acid and low yield

Active Publication Date: 2020-05-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the existing microbial fermentation method still has certain defects, wherein low yield is one of the defects that limit the industrialized production process of L-glutamic acid

Method used

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  • Recombinant corynebacterium glutamicum and application thereof in producing L-glutamic acid
  • Recombinant corynebacterium glutamicum and application thereof in producing L-glutamic acid
  • Recombinant corynebacterium glutamicum and application thereof in producing L-glutamic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Construction and selection of knockout plasmids

[0057] See the build process figure 1 ,Specific steps are as follows:

[0058] (1) Dip the bacteria solution of Corynebacterium glutamicum (Corynebacterium glutamicum) E01 and Corynebacterium glutamicum (Corynebacterium glutamicum) G01 from the glycerol tube and inoculate them into the seed medium, and inoculate them under the conditions of 30°C and 180r / min Cultivate under low temperature for 18 hours to the logarithmic growth phase to obtain the culture solution; centrifuge the culture solution at -4°C and 6000r / min for 10 minutes to collect the bacteria; freeze the bacteria in liquid nitrogen for 10 minutes, store them in a -80°C refrigerator, and send them to Suzhou Jinweizhi Co., Ltd. carried out total RNA extraction, total RNA-seq and resequencing analysis; concentrated analysis of genes related to the central metabolic pathway of Corynebacterium glutamicum (Corynebacterium glutamicum) E01 and Corynebac...

Embodiment 2

[0082] Example 2: Construction and fermentation of recombinant Corynebacterium glutamicum C. glutamicum DL01

[0083] Specific steps are as follows:

[0084] The knockout plasmid pFSC-dCas9-S3 obtained in Example 1 was electroporated into Corynebacterium glutamicum G01 to obtain the transformation product; the transformation product was spread on LBG medium supplemented with chloramphenicol, and placed in a 30°C constant temperature incubator Inverted culture for 36 hours to obtain transformants; Streak the transformants on LBG medium supplemented with chloramphenicol, and culture them upside down in a constant temperature incubator at 30°C for 36 hours to obtain a single colony; use dCas9-F and sgRNA-R as primers Carry out colony PCR verification (verification results see Figure 4 ), the verification is correct to obtain recombinant Corynebacterium glutamicum C. glutamicum DL01; with Corynebacterium glutamicum (Corynebacterium glutamicum) G01 as a control, a single colony o...

Embodiment 3

[0086] Example 3: Construction and fermentation of recombinant Corynebacterium glutamicum C. glutamicum G01 / pDXW-10-ppc

[0087] Specific steps are as follows:

[0088] Using the genome of Corynebacterium glutamicum G01 as a template and using pDXW-10-ppc-F and pDXW-10-ppc-R as primers for PCR amplification, the nucleotide sequence such as SEQ ID No. The gene ppc encoding phosphoenol pyruvate carboxylase shown in 4; the gene ppc encoding phosphoenol pyruvate carboxylase and the pDXW-10 plasmid are digested with restriction enzymes EcoRI and HindIII Ligation was performed to obtain the ligation product; the ligation product was transformed into Escherichia coli (Escherichia coli) JM109 to obtain the transformation product; the transformation product was coated on LB solid medium (containing 50 μg·mL -1 Kanamycin), cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick the transformants and inoculate them into LB liquid med...

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Abstract

The invention discloses recombinant corynebacterium glutamicum and application thereof in producing L-glutamic acid and belongs to the technical field of biologics. The invention provides the recombinant C.glutamicum DL01 / pDXW-10-ppc capable of producing L-glutamic acid with a high yield, the recombinant C.glutamicum DL01 / pDXW-10-ppc is inoculated into a 5-L fermentation tank for fermentation for48 hours, then the yield of the L-glutamic acid in a fermentation broth can be as high as 136.09+ / -5.53g / L, the sugar acid conversion rate is as high as 58.9%, and the yield and the sugar acid conversion rate are respectively increased by 45.5% and 13.7% when being compared with those of wild Corynebacterium glutamicum G01.

Description

technical field [0001] The invention relates to a recombinant Corynebacterium glutamicum and its application in producing L-glutamic acid, belonging to the field of biotechnology. Background technique [0002] L-Glutamic acid is an important amino acid, which is widely used in many fields. For example, L-glutamic acid has a strong umami taste and can be used as a flavoring agent in the food industry; L-glutamic acid can be used to synthesize surfactants and has important applications in the field of cosmetics; L-glutamic acid is used in the human body After absorption, it is easy to form glutamine with blood ammonia, which can relieve the toxic effect of ammonia in the metabolic process. It can be used as an auxiliary drug for patients with liver diseases and has important applications in the medical field. It is effective in treating concussions or nerve damage, epilepsy, and mentally handicapped children. [0003] The production methods of L-glutamic acid mainly include ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/77C12P13/14C12R1/15
CPCC12N9/88C12Y401/01031C12N15/77C12P13/14C12N1/20
Inventor 饶志明乔郅钠徐美娟龙梦飞杨套伟张显邵明龙
Owner JIANGNAN UNIV
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