Construction method of bacillus subtilis engineering bacteria for high yielding of alanine

A technology of Bacillus subtilis and construction method, applied in the field of microbiology

Inactive Publication Date: 2015-05-06
ANHUI UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Based on this, the production of alanine can be improved by increasing the carbon source flowing to alanine by knocking out the lactate dehydrogenase gene (ldh) and the acetate kinase gene (ackA), but the knockout of the gene may have a negative effect on the strain itself. have an effect on growth

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  • Construction method of bacillus subtilis engineering bacteria for high yielding of alanine
  • Construction method of bacillus subtilis engineering bacteria for high yielding of alanine
  • Construction method of bacillus subtilis engineering bacteria for high yielding of alanine

Examples

Experimental program
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Embodiment Construction

[0037] (1) The methods used in the following examples are conventional methods unless otherwise specified, and the B. subtilis IBL23 used is the serial number of the model strain B. subtilis 168 in our laboratory.

[0038] 1. Construction of lactate dehydrogenase single gene knockout mutant B. subtilis IBL23-ldh

[0039] The build method includes the following steps:

[0040] 1.1B.subtilis IBL23 genome extraction

[0041] Genomic DNA of B. subtilis IBL23 was extracted using Shanghai Sangon SK8255 kit

[0042] 1). Take 1ml of B. subtilis IBL23 cultured overnight, add it to a 1.5ml EP tube, centrifuge at room temperature for 1 minute at 8000 rpm, discard the supernatant thoroughly, and collect the bacteria.

[0043] 2). Add 180 μl of 20 mg / ml lysozyme solution to resuspend the bacteria, bathe in 37°C water for 0.5-1 hour, and invert the EP tube every 5-10 minutes while in the water bath.

[0044] 3). Add 20 μl of proteinase K solution and oscillate with a fast mixer to mix. ...

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Abstract

The invention discloses a preparation method of bacillus subtilis engineering bacteria for high yielding of alanine, wherein the method comprises that alanine-synthesized branched metabolism genes L-lactate dehydrogenase gene ldh and acetate kinase gene ackA on a B.subtilis 168 chromosome are knocked out by using a homologous recombination method, and a mutant strain is obtained. The single-gene mutant strain has the preservation number of IBL23-ldh, the construction of the mutant strain is achieved by knocking out a part of the lactate dehydrogenase gene of bacillus subtilis; a double-gene mutant strain is obtained by again knocking out the acetate kinase gene based on the single-gene mutant strain and has the number for IBL23-ldh / ackA.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for constructing a highly alanine-producing Bacillus subtilis engineering bacterium. Background technique [0002] L-alanine is a non-essential aliphatic non-polar amino acid, but it is the amino acid with the highest content in human blood. Alanine plays an important role in the metabolism of organisms and is used in food industry and medicine. is of great significance. It was not until the 1980s that industrial production was formed in Japan. The preparation of L-alanine was from the initial proteolysis extraction method and fermentation method to the now popular enzymatic method. Enzymatic conversion is obtained by catalyzing L-aspartic acid by microorganisms (such as pseudomonas dacunhae, etc.) rich in L-aspartic acid-B-decarboxylase activity. Among them, immobilized cell method is mostly used in Japan, while my country uses Free intact cell method. Many microorganism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/20C12P13/06C12R1/125
Inventor 童望宇于振海谢球
Owner ANHUI UNIVERSITY
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