Improvement of L-arginine yield of corynebacterium crenatum by enhancement of transport protein LysE expression

A technology of Corynebacterium blunt-toothed and transporter, which is applied in the field of genetic engineering, and can solve the problems that have not yet been reported on the arginine transporter of Corynebacterium blunt-toothed.

Inactive Publication Date: 2012-10-24
JIANGNAN UNIV
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  • Improvement of L-arginine yield of corynebacterium crenatum by enhancement of transport protein LysE expression
  • Improvement of L-arginine yield of corynebacterium crenatum by enhancement of transport protein LysE expression
  • Improvement of L-arginine yield of corynebacterium crenatum by enhancement of transport protein LysE expression

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Embodiment 1

[0026] Example 1: Amplification of the target gene and construction of recombinant Corynebacterium blunt-toothed

[0027] Using the C. crenatum SYPA5-5 chromosomal genome as a template, using CclysE F BamHI and CclysE R Sal I as primers, PCR amplified to obtain the lysE gene containing two restriction sites of BamHI and Sal I, which was connected to pMD18-T Vector, Construct the cloning vector pMD18-T-CclysE(BamHI+Sal I), transform the cloning vector pMD18-T-CclysE(BamHI+SalI) into the recipient Escherichia coli, spread on the LB agar plate containing Amp, and culture at 37°C After overnight, positive transformants were randomly picked and identified by enzyme digestion and sent to Shanghai Sangon for sequencing and sequence analysis. Double digestion with BamHI and Sal I obtained the lysE gene containing two restriction sites of BamHI and Sal I. The enzyme-digested shuttle expression vector pJC-tac was ligated to obtain the recombinant shuttle expression vector pJC-tac-lysE, ...

Embodiment 2

[0033] Example 2: Detection of L-arginine secretion and analysis of extracellular and extracellular L-arginine content in the original and recombinant bacteria of Corynebacterium blunt tooth

[0034] 10 5 Each / mL cell concentration was mixed into E.coli BL21L-arginine-deficient bacterial strain to make detection plate, and the corynebacterium bacilli constructed in the present invention pJC-tac-lysE / C.crenatum 5-5 (numbering 4) and the original bacteria C. crenatum 5-5 (No. 3) were planted on the plate with the same amount, cultured at 30°C for 3-5 days, and evenly sprayed sterile 1 μg / mL TTC solution on the ultra-clean workbench (membrane filtration sterilization), Place at 37°C for 1-2h, and observe the color development on the plate. The size of the red circle around the strain is determined by the growth of L-arginine-deficient E. coli, and the growth of E. coli is positively correlated with the secretion of L-arginine. The result is as Figure 5 , the diameter of the r...

Embodiment 3

[0037] Example 3: Fermentation performance verification of Corynebacterium blunt tooth original bacteria and recombinant bacteria

[0038] Based on the result verification of example 2, carry out 250mL shaking flask fermentation experiment to recombinant bacterium pJC-tac-lysE / C.crenatum 5-5 and original bacterium C.crenatum 5-5, from fermentation angle to the expression of lysE gene in Corynebacterium blunt tooth The effect of overexpression on improving the production of L-arginine was verified. The speed of the reciprocating shaker is 200r / min, 30°C, fermented for 96 hours, and samples are taken every 12 hours. The growth of bacteria, the consumption of glucose, and the content of extracellular L-arginine in each group of samples at each fermentation time point (the extracellular content is the yield) and the yield were tracked and measured for comparison and analysis, and the contents of various amino acids in the fermentation broth fermented by each strain for 96 hours we...

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Abstract

Endocellular secretion of L-arginine to the exterior of cells depends on transport protein LysE. According to the invention, corynebacterium crenatum is used as a research bacterial strain for the first time; a shuttle expression plasmid pJC-tac-lysE carrying a lysE gene of its own is constructed and is electrically transferred to the corynebacterium crenatum so as to obtain a recombinant corynebacterium crenatum with enhanced lysE gene expression. The original bacterium and the recombinant bacterium are qualitatively detected through plate color development and are cultured by a basal medium so as to examine the L-arginine transport status; over expression of the L-arginine transport protein LysE in the corynebacterium crenatum is verified, which indicates that the enhancement of transport protein LysE expression can reduce the intracellular L-arginine concentration to a certain extent and improve the L-arginine yield. The original bacterium and the recombinant bacterium are subject to a fermentation experiment in a 250-mL shake flask, and results show that the L-arginine yield of the recombinant bacterium is increased by about 12%; the maximal yield is increased by 11.1%; and the fermentation period is shortened.

Description

technical field [0001] The invention relates to improving the production of L-arginine by strengthening the expression of lysE gene in corynebacterium bacilli, and belongs to the technical field of genetic engineering. 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] Increasing the expression of enzymes in the synthesis pathway is one of the most important strategies to increase the metabolic flux of the L-arginine synthesis pathway and ultimately increase its yield. This is also the main ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/10C12R1/15
Inventor 许正宏饶志明徐美娟张博慧杨娟刘巧利窦文芳
Owner JIANGNAN UNIV
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