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A kind of anti-acid stress recombinant lactic acid bacteria and its construction method

A technology for recombining lactic acid bacteria and lactic acid bacteria, applied in the field of bioengineering, can solve the problems of low success rate, weakening existing metabolic pathways, and increasing production costs.

Active Publication Date: 2020-01-07
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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  • A kind of anti-acid stress recombinant lactic acid bacteria and its construction method
  • A kind of anti-acid stress recombinant lactic acid bacteria and its construction method
  • A kind of anti-acid stress recombinant lactic acid bacteria and its construction method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Construction of recombinant strain

[0025] Obtain the gene sequence of LtrC as shown in SEQ ID NO.2 from L.lactis NZ9000 in the NCBI database, and clone it into the expression plasmid pNZ8148 of Lactococcus lactis to obtain the recombinant plasmid pNZ8148 / LtrC, and then electrotransform it into The recombinant strain L.lactis NZ9000 (pNZ8148 / LtrC) was obtained from the host strain L.lactis NZ9000.

[0026] details as follows:

[0027] The primers lrC-F and lrC-R shown in SEQ ID NO. 3 and SEQ ID NO. 4 were designed according to the gene sequence of LtrC (Table 1), and PCR amplification was performed using the genome of L.lactis NZ9000 as a template to obtain SEQ ID NO. The gene fragment shown in ID NO.2. The PCR product and vector pNZ8148 were digested with Nco Ⅰ and Hind Ⅲ, respectively, and the digested product was purified and then connected. The ligation product was transformed into E. coli MC1061 (commercial strain) competent, and positive clones were screene...

Embodiment 2

[0031] Example 2 Growth performance test of LtrC overexpressing strain

[0032] For the investigation of the growth of the strains when overexpressing LtrC, the strains L.lactis NZ9000 (pNZ8148 / LtrC) and L.lactis NZ9000 (pNZ8148) (control) were inoculated into GM17 liquid medium supplemented with 10μg / mL chloramphenicol ( 1mL) for activation 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 LtrC protein. Take time as the abscissa, OD 600 The value is the ordinate, and the growth curve is drawn.

[0033] The result is figure 2 Shown. After growth performance test analysis, the biomass of the recombinant strain and the control strain ...

Embodiment 3

[0034] Example 3 Tolerance test under acid stress conditions

[0035] For the analysis test to investigate the acid tolerance of the strains, the survival rates of the recombinant strains and the control strains under the condition of pH 4.0 were determined.

[0036] 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) ), the coercion is 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.

[0037] According to the tolerance experiment analysis, the survival rate of the recombinant strain L.lactis NZ90...

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Abstract

The invention discloses acid stress resistant recombinant lactic acid bacteria and a construction method thereof, and belongs to the field of bioengineering technology. By overexpression of LtrC genederived from L.lactis NZ9000 in L.lactis NZ9000, the recombinant L.lactis NZ9000 (pNZ8148 / LtrC) with acid stress resistance remarkably enhanced is obtained. By stress for 3 h at pH 4.0, survival rateof the recombinant strain is 3.6 times higher than survival rate of a contrast. The invention also provides a method for raising acid stress resistance. The method of the invention has good industrialapplication value.

Description

Technical field [0001] The invention relates to an acid-resistant recombinant lactic acid bacterium and a construction method thereof, 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 neutralizers 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 enviro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C07K14/195C12R1/46
CPCC07K14/195
Inventor 张娟陈坚堵国成朱政明
Owner JIANGNAN UNIV
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