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Anti-acid-stress component and application thereof

A resistance, amino acid technology, applied in the field of bioengineering, can solve problems such as low efficiency, weakening existing metabolic pathways, and low success rate

Active Publication Date: 2017-10-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for improving the acid stress tolerance of lactic acid bacteria mainly contains at present: (1) mutagenesis breeding, this method has the characteristics such as easy, type is various, but workload is big, efficient is its main shortcoming; (2) biochemical engineering strategy, It has been reported that exogenous aspartic acid has been used to improve the acid stress tolerance of lactic acid bacteria, but the use of this method has caused an increase in production costs; (3) Metabolic engineering strategies, currently using metabolic engineering strategies to improve the environmental stress tolerance of lactic acid bacteria The methods mainly include constructing new metabolic pathways, expanding existing metabolic pathways and weakening existing metabolic pathways.
The above methods either have cost issues or low success rates

Method used

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  • Anti-acid-stress component and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of recombinant strain

[0031] Obtain the gene sequence of recT-A76 as shown in SEQ ID NO.3 from L.lactis A76 in the NCBI database, and clone it into the expression plasmid pNZ8148 of Lactococcus lactis to obtain the recombinant plasmid pNZ8148 / RecT-A76. It was electrotransformed into the host bacteria L.lactis NZ9000 to obtain the recombinant strain L.lactis NZ9000 (pNZ8148 / RecT-A76); the recT-MG1655 shown in SEQ ID NO.4 was obtained from E.coli MG1655 in the NCBI database The gene sequence was cloned into the expression plasmid pNZ8148 to obtain the recombinant plasmid pNZ8148 / RecT-MG1655, and then electrotransformed into the host bacteria L.lactis NZ9000 to obtain the recombinant strain L.lactis NZ9000 (pNZ8148 / RecT-MG1655).

[0032] details as follows:

[0033] Design primers recT-A76-BF, recT-A76-BR, recT-MG1655- as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQID NO.8 according to the gene sequence of recT. BF, recT-MG1655-BR (Table 1), usin...

Embodiment 2

[0037] Example 2 Growth performance test of strains overexpressing RecT protein

[0038] For investigating the growth of strains when overexpressing RecT protein, the strains L.lactis NZ9000 (pNZ8148 / RecT-A76), L.lactis NZ9000 (pNZ8148 / RecT-MG1655) and L.lactis NZ9000 (pNZ8148) (control) were inoculated It was activated in GM17 liquid medium (1 mL) supplemented with 10 μg / mL chloramphenicol, and placed in a 30°C incubator for static culture overnight. Then, the seed solution was transferred to fresh chloramphenicol (10 μg / mL) GM17 liquid medium with an inoculum amount of 2%, and the culture was statically cultured at 30°C. Sampling was taken every 2 hours to determine the OD value at a wavelength of 600nm. Cultivate to OD 600 At 0.4, 10ng / mL nisin was added to induce the expression of RecT protein. Take time as the abscissa, OD 600 The value is the ordinate, and the growth curve is drawn.

[0039] The result is figure 2 Shown. The growth performance test analysis showed that t...

Embodiment 3

[0040] Example 3 Tolerance test under acid stress conditions

[0041] For the analysis test to investigate the acid tolerance of the strain, the survival rate of the recombinant strain and the control strain under pH 4.0 conditions were determined.

[0042] The specific operation method is as follows: the strain is induced and cultured for 6 hours, the cells are collected by centrifugation, washed twice with 0.85% saline and resuspended in an equal volume of fresh GM17 (containing 10 μg / mL chloramphenicol) with pH 4.0 (adjusted by lactic acid) In, coercion at different times. The strained bacteria suspension was washed twice and then resuspended in an equal volume of normal saline. 10 μL of the resuspension was taken, diluted with different gradients and planted on GM17 chloramphenicol plates to determine the number of viable bacteria and survival rate.

[0043] According to the tolerance experiment analysis, after 3 hours of stress in GM17 at pH 4.0, the survival rates of recombina...

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Abstract

The invention discloses an anti-acid-stress component and application thereof, belonging to the field of bioengineering technology. According to the invention, two recombinant Lactococcus lactis strains with substantially improved resistance to acid stress, i.e., L. lactis NZ9000 (pNZ8148 / RecT-A76) and L. lactis NZ9000 (pNZ8148 / RecT-MG1655), are obtained by overexpression of recT genes, originated from Lactococcus lactis L. lactis A76 and Escherichia coli E. coli MG1655, in Lactococcus lactis NZ9000, respectively. After the two recombinant Lactococcus lactis strains suffer from stress in an environment with a pH value of 4.0 for 3h, the survival rates of the two strains are 10.5 times and 1.4 times the survival rates of control strains, respectively; and after the two strains suffer from stress caused by 20% (v / v) ethanol for 6 h, the survival rates of the two strains are 3.7 times and 2.1 times the survival rates of control strains, respectively. The invention also provides a method for improving resistance to acid stress. The method has good industrial application value.

Description

Technical field [0001] The invention relates to an acid stress resistant component and its application, and belongs to the technical field of biological engineering. Background technique [0002] When lactic acid bacteria are used for industrial production, in the fermentation process, along with the metabolic growth of the bacteria, acidic substances are also produced and accumulated, causing the cells to face severe acid stress. In order to maintain the stability of fermentation production and improve production efficiency, the industry usually maintains the pH in a stable range by adding exogenous neutralizing agents during the fermentation process. For example, by adding alkaline substances (ammonia or NaOH) to control the pH value of the fermentation environment. However, the addition of alkaline substances often leads to the accumulation of by-products. The salts formed in the by-products will once again cause the cells to be in a hypertonic environment, resulting in the ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C07K14/245C07K14/315A23L33/135A23K10/16C12R1/46
CPCA23K10/16A23L33/135C07K14/245C07K14/315
Inventor 张娟陈坚堵国成吉晓梅朱政明
Owner JIANGNAN UNIV
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