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Acid stress resistant recombinant lactic acid bacteria and construction method thereof

A technology for recombining lactic acid bacteria and Lactococcus lactis, applied in the field of bioengineering, can solve problems such as low success rate, heavy workload, and low efficiency

Active Publication Date: 2018-03-23
JIANGNAN UNIV
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  • 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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  • Acid stress resistant recombinant lactic acid bacteria and construction method thereof
  • Acid stress resistant recombinant lactic acid bacteria and construction method thereof
  • Acid stress resistant recombinant lactic acid bacteria and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construction of embodiment 1 recombinant strain

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

[0026] details as follows:

[0027] According to the gene sequence of LtrC, primers lrC-F and lrC-R (table 1) shown in SEQ ID NO.3 and SEQ ID NO.4 were designed respectively, and the genome of L.lactis NZ9000 was used as template PCR amplification to obtain SEQ ID NO. The gene fragment shown in ID NO.2. The PCR product and the vector pNZ8148 were double-digested with Nco I and Hind III, respectively, and the digested products were purified and ligated. The ligation product was transformed into Escherichia coli MC1061 (commercial strain) competent, positive clon...

Embodiment 2

[0031] The growth performance test of embodiment 2 overexpression LtrC bacterial strain

[0032] To investigate the growth of bacterial strains when overexpressing LtrC, bacterial strains L.lactis NZ9000 (pNZ8148 / LtrC) and L.lactis NZ9000 (pNZ8148) (control) were inoculated in GM17 liquid medium added with 10 μg / mL chloramphenicol ( 1 mL) for activation, and placed in a 30°C incubator for static culture overnight. The seed solution was then transferred to fresh chloramphenicol (10 μg / mL) GM17 liquid medium with a 2% inoculation amount, and cultured statically at 30°C. Samples were taken every 2 hours, and the OD value at a wavelength of 600nm was measured. Grow to OD 600 At 0.4, 10ng / mL nisin was added to induce the expression of LtrC protein. Taking time as the abscissa, OD 600 The value is the vertical axis, and the growth curve is drawn.

[0033] The result is as figure 2 shown. The growth performance test analysis showed that the biomass of the recombinant strain w...

Embodiment 3

[0034] Tolerance test under the acid stress condition of embodiment 3

[0035] For the analysis of the tolerance of the investigated strains to acid, the survival rates of the recombinant strains and the control strains at pH 4.0 were measured.

[0036] The specific operation method is as follows: the strain was induced and cultured for 6 h, the cells were collected by centrifugation, washed twice with 0.85% normal saline, and then resuspended in an equal volume of fresh GM17 (containing 10 μg / mL chloramphenicol) at pH 4.0 (adjusted by lactic acid). ), the coercion time is different. After the stress, the bacterial suspension was washed twice and resuspended in an equal volume of normal saline, and 10 μL of the resuspension was taken, diluted with different gradients and planted on a GM17 chloramphenicol plate to determine the number of viable bacteria and the survival rate.

[0037] According to the analysis of the tolerance test, the survival rate of the recombinant strain ...

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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 stress-resistant recombinant lactic acid bacterium and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] When lactic acid bacteria are used for industrial production, during 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, the pH value of the fermentation environment is controlled by adding alkaline substances (ammonia or NaOH). However, the addition of alkaline substances often leads to the accumulation of by-products. The salts formed in the by-products will again cause the cells to be in a hypertonic en...

Claims

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Application Information

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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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