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A method of using ion transporter to promote the synthesis of L-arginine by Corynebacterium bacillus

A technology of Corynebacterium blunt tooth and antiporter, which is applied in the field of bioengineering to achieve the effect of increasing production

Active Publication Date: 2022-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This strain does not have L-arginine deaminase, which makes the L-arginine produced in the cell not degraded or degraded to a limited extent, and has a highly efficient promotion effect on the synthesis of L-arginine, so its mutation strain is widely used in domestic amino acid production, but the research on its genetic background is still blank

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  • A method of using ion transporter to promote the synthesis of L-arginine by Corynebacterium bacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Knockout of ion transporters Mrp1A and CTAP1

[0026] With Corynebacterium bacillus SYPA5-5 genome as template, utilize primer pair Δmrp1-1 / Δmrp1-2 (nucleotide sequence as shown in SEQ ID NO.5 / 6) and Δmrp1-3 / Δmrp1-4 (nucleotide The sequence is shown in SEQ ID NO.7 / 8) to amplify the upstream and downstream fragments of the mrp1 gene by 500 bp each. After recovery, use the above and downstream fragments as templates, and use mrp1-1 (nucleotide sequence as shown in SEQ ID NO.5) and mrp1-4 (nucleotide sequence as shown in SEQ ID NO.8) primer fusion PCR amplification The missing fragment of the mrp1 gene was obtained. Purify and recover the PCR product to obtain the mrp1 gene deletion fragment, purify and recover the digested product of pK18mobSacB plasmid, and use homologous recombinase for connection. The ligation product was transformed into E.coli JM109 competent by heat shock, and the transformants were selected by culture on LB plates containing kanamycin ...

Embodiment 2

[0029] Example 2: Overexpression of the ion transporters Mrp1A and CTAP1

[0030] With Corynebacterium bacillus SYPA5-5 genome as template, utilize primer pair 10-mrp1ctap1-1 / 2 (nucleotide sequence as shown in SEQ ID NO.13 / 14) and 10-mrp1ctap1-3 / 4 (nucleotide The sequence is shown in SEQ ID NO.15 / 16) to amplify the upstream mrp1 and downstream ctap1 gene fragments respectively. After recovery, the above and downstream fragments were used as templates, and the tandem gene mrp1ctap1 was amplified by fusion PCR using the primer pair 10-mrp1ctap1-1 / 4. Purify and recover the PCR product to obtain the mrp1ctap1 gene fragment, purify and recover the digested product of the pDXW-10 plasmid, and use homologous recombinase for connection. The ligation product was transformed into E.coli BL21 competent by heat shock, and the transformants were screened by kanamycin resistance plate culture, and the plasmid was extracted and digested, PCR and sequencing were verified. The verification w...

Embodiment 3

[0031] Example 3: Mrp1A and CTAP1 on intracellular [Na + ] and [K + ]Impact

[0032] Take 1mL of bacterial liquid, centrifuge at 8000r / min for 2min, discard the supernatant, wash the bacterial cells with distilled water 3 times, dry at 60°C, weigh the dry cell weight, 1OD 562 =0.375g / L dry weight of cells. Take 1mL of bacteria, digest completely with 1mL of concentrated nitric acid until clarified, and then dilute 100 times. The diluted liquid was filtered through a membrane, and the recombinant strain [Na + ] / [K + ] content. Calibrate with ultrapure water.

[0033] At pH 7.0, the Na of the 5-5Δmrp1Δctap1 mutant compared + concentration increased significantly, while K + The concentration is lower. This is due to the absence of ion transporters leading to intracellular Na + cannot be transported out of the cell, intracellular Na + Accumulation leads to a significant increase in concentration, and the cell needs to adjust the intracellular ionic strength, so that the...

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Abstract

The invention discloses a method for using ion transporters to promote the synthesis of L-arginine by Corynebacterium blunt-toothed, and specifically discloses a method for overexpressing monovalent cations / H + The invention relates to a method for increasing the production of L-arginine by antiporter protein Mrp1A and cation transporter ATPase CTAP1, belonging to the technical field of bioengineering. The present invention has successfully realized the knockout and overexpression of genes mrp1 and ctap1, through the intracellular [Na + ] and [K + ] to clarify the regulatory role of ion transporters in ion concentration, pH homeostasis and osmotic pressure. A 5L fermenter batch fermentation strategy was adopted to optimize the fermentation conditions. Finally, the recombinant Corynebacterium blunt-toothed 5-5 (mrp1ctap1) was fermented for 96 hours, and the L-arginine output of the recombinant bacteria reached 65.3g / L, with a yield of 0.395g / L. g, 39.1% higher than Corynebacterium blunt tooth SYPA5‑5.

Description

technical field [0001] The invention discloses a method for using ion transporters to promote the synthesis of L-arginine by Corynebacterium blunt tooth, and specifically discloses a method for overexpressing monovalent cations / H + The invention relates to a method for increasing the production of L-arginine by antiporter protein Mrp1A and cation transport ATPase CTAP1, belonging to the technical field of bioengineering. Background technique [0002] L-Arginine (C 6 h 14 N 4 o 2 ) is a natural basic amino acid, which is widely used in food additives, feed and pharmaceutical industries and is beneficial to human growth and development. L-arginine has a variety of physiological functions and effects, and is widely used in human production and life. It is one of the hot spots in the research and development of the amino acid industry. In the process of L-arginine synthesis, ion transport provides a suitable intracellular pH environment for the fermentation of Corynebacteri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/10C12R1/15
CPCC12N9/14C07K14/34C12N15/77C12P13/10
Inventor 饶志明徐美娟刘晶张显杨套伟邵明龙
Owner JIANGNAN UNIV
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