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Lactic acid bacteria engineering strain with improved acid stress resistance

A technology of lactic acid bacteria and engineering bacteria, applied in the fields of genetic engineering and microbial engineering, can solve the problems of affecting the growth and metabolism of bacteria, accumulation and weakening of by-products, etc., and achieve the improvement of amino acid stress resistance, acid stress resistance and lactic acid. The effect of increasing resistance

Active Publication Date: 2019-03-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the addition of alkaline substances often leads to the accumulation of by-products, and the salts formed in the by-products will once again lead to a hypertonic environment for cells, resulting in osmotic stress, which will affect the growth and metabolism of bacteria again
[0008] At present, the method that improves the acid stress resistance such as lactic acid, acetic acid of lactic acid bacteria then mainly contains: (1) mutagenesis breeding, this method has characteristics such as easy, various types, but workload is big, efficient is its main shortcoming; (2) ) biochemical engineering strategy, it has been reported that exogenous aspartic acid has been added 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 strategy, currently using metabolic engineering Strategies to improve the environmental stress of lactic acid bacteria mainly include constructing new metabolic pathways, expanding existing metabolic pathways and weakening existing metabolic pathways. However, this method has the problems of high cost and low success rate.

Method used

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  • Lactic acid bacteria engineering strain with improved acid stress resistance
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  • Lactic acid bacteria engineering strain with improved acid stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of recombinant strains

[0054] Specific steps are as follows:

[0055] (1) Obtain the purC gene sequence shown in SEQ ID NO.1 from the NCBI database (the gene encoding phosphoribosylaminoimidazole-succinamide synthetase PurC, which participates in the IMP biosynthetic pathway in purine metabolism, which itself is Part of purine metabolism, regulation of purine metabolism can alleviate the intrusion of abiotic stress to a certain extent), according to the gene sequence design the primers shown in Table 1;

[0056] (2) Using the genome of L.lactis NZ9000 as a template, and using the primers in Table 1 to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification;

[0057] (3) The PCR product and the vector pNZ8148 were respectively digested with the restriction enzymes in Table 1, and the digested product was purified and then connected;

[0058] (4) The ligation product was transformed into E. coli MC1061 (commercial strain) competent, and positiv...

Embodiment 2

[0062] Example 2: Growth performance test of recombinant strain

[0063] Specific steps are as follows:

[0064] (1) The strain L lactis NZ9000 (pNZ8148) (control) and the strain L lactis NZ9000 (pNZ8148 / purC) obtained in Example 1 were respectively inoculated into GM17 liquid medium supplemented with 10 μg / mL chloramphenicol to activate them. Cultivate overnight in a 30°C incubator;

[0065] (2) Transfer the seed solution obtained above to fresh chloramphenicol (10μg / mL) GM17 liquid medium with an inoculum of 2% respectively, and cultivate it at 30°C;

[0066] (3) During the cultivation process, take samples at regular intervals to determine the OD value at 600nm wavelength;

[0067] (4) Cultivate to OD 600 At 0.4, 10ng / mL nisin was added to induce the expression of transporter, with time as abscissa, OD 600 The value is the ordinate, and the growth curve is drawn (the drawn growth curve is as figure 2 Shown).

[0068] The result is figure 2 As shown, after growth performance test a...

Embodiment 3

[0069] Example 3: Tolerance test of recombinant strains under acid stress conditions

[0070] Specific steps are as follows:

[0071] The strain L lactis NZ9000 (pNZ8148) (control) and the strain L lactis NZ9000 (pNZ8148 / purC) obtained in Example 1 were induced and cultured for 6 hours. The cells were collected by centrifugation, washed twice with 0.85% physiological saline and resuspended in an equal volume In fresh pH 4.0 (adjusted by lactic acid) GM17 (containing 10μg / mL chloramphenicol), stress for different times; wash the strained bacterial suspension twice, then resuspend in an equal volume of normal saline, take 10μL and resuspend Solution, dilute different gradient spots and plant on GM17 chloramphenicol plates to determine the number of viable bacteria and survival rate (the results are as image 3 Shown);

[0072] Survival rate = (N / N 0 )×100%;

[0073] Where N 0 Is the number of viable colonies on the plate without acid stress treatment; N is the number of viable colonies...

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Abstract

The invention discloses lactic acid bacteria engineering bacteria with improved acid stress resistance, and belongs to the genetic engineering and microbial engineering technical fields. The lactic acid bacteria engineering bacteria which can be widely applied in preparation of food, medicines, feeds and chemicals are successfully constructed by taking a gene encoding phosphoribosylaminimidazole-succinimide synthetase PurC as a target gene and lactic acid bacteria as an expression host. The acid stress resistance of the lactic acid bacteria engineering bacteria is significantly improved, and the maximum acid stress resistance is improved by 83.2 times than that of wild strains.

Description

Technical field [0001] The invention relates to an engineered lactic acid bacteria strain with improved acid stress resistance, belonging to the technical field of genetic engineering and microbial engineering. Background technique [0002] Lactic acid bacteria is a general term for bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. This type of bacteria is extremely widespread in nature and has a rich diversity of species. They are not only ideal materials for studying classification, biochemistry, genetics, molecular biology and genetic engineering, but also have important academic value in theory, but also in important industrial fields closely related to human life such as food, medicine, feed, and fine chemicals. It also has important application value. [0003] However, in the process of industrial fermentation and production of lactic acid bacteria and the role of probiotics in the human gastrointestinal system, they inevitably face vari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
CPCC12N9/93C12N15/746C12Y603/02006
Inventor 张娟杨佩珊刘为佳陈坚堵国成
Owner JIANGNAN UNIV
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