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Lactic acid bacteria engineering bacterium with improved acid stress resistance and application thereof

A kind of technology of lactic acid bacteria and engineering bacteria

Active Publication Date: 2019-01-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 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
[0007] 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 bacterium with improved acid stress resistance and application thereof
  • Lactic acid bacteria engineering bacterium with improved acid stress resistance and application thereof
  • Lactic acid bacteria engineering bacterium with improved acid stress resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Construction of recombinant bacterial strain

[0058] Specific steps are as follows:

[0059] (1) Obtain the zitP gene sequence shown in SEQ ID NO.1, the zitQ gene sequence shown in SEQ ID NO.3, the bglF gene sequence shown in SEQ ID NO.4, and the bglF gene sequence shown in SEQ ID NO.4 from the NCBI database. For the ganP gene sequence shown in ID NO.5, the primers shown in Table 1 were designed according to the gene sequence;

[0060] (2) Using the genome of L.lactis NZ9000 as a template, the primers in Table 1 were amplified by PCR to obtain SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5. gene fragments;

[0061] (3) The PCR product and the carrier pNZ8148 were double-digested with the restriction endonucleases in Table 1, and the digested products were purified and ligated;

[0062] (4) Transform the ligation product into Escherichia coli MC1061 (commercialized strain) competent, screen positive clones on a chloramphenicol plate, verify by c...

Embodiment 2

[0066] Embodiment 2: the growth performance test of recombinant bacterial strain

[0067] Specific steps are as follows:

[0068] (1) The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strain L lactis NZ9000 (pNZ8148 / zitP) obtained in Example 1, L lactis NZ9000 (pNZ8148 / zitQ), L lactis NZ9000 (pNZ8148 / bglF), L lactis NZ9000 (pNZ8148 / ganP) were respectively inoculated in GM17 liquid medium supplemented with 10 μg / mL chloramphenicol for activation, and placed in a 30°C incubator for static culture overnight;

[0069] (2) Transfer the above-obtained seed solution to fresh chloramphenicol (10 μg / mL) GM17 liquid medium with an inoculum size of 2%, and culture it statically at 30° C.;

[0070] (3) During the culturing process, samples were taken at regular intervals to measure the OD value at a wavelength of 600nm;

[0071] (4) Cultivate to OD 600 Add 10ng / mL nisin at 0.4 to induce the expression of the transporter, take time as the abscissa, OD 600 The ...

Embodiment 3

[0074] Example 3: Tolerance test of recombinant strains under lactic acid stress conditions

[0075] Specific steps are as follows:

[0076] The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strains L lactis NZ9000 (pNZ8148 / zitP) and lactis NZ9000 (pNZ8148 / zitQ) obtained in Example 1 were respectively induced and cultured for 6 h, and the cells were collected by centrifugation, and washed with 0.85% normal saline for two times. Resuspend in an equal volume of fresh GM17 (containing chloramphenicol 10 μg / mL) at pH 4.0 (adjusted by lactic acid) for the second time, and stress for different times; wash the stressed bacterial suspension twice and resuspend in an equal volume of In normal saline, take 10 μ L of the resuspension, dilute different gradients, and plant on the GM17 chloramphenicol plate to determine the number of viable bacteria and the survival rate (the results are as follows: Figure 7 and Figure 8 shown).

[0077] Such as Figure 7 and...

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Abstract

The invention discloses a lactic acid bacteria engineering bacterium with improved acid stress resistance and an application thereof, belonging to the technical field of genetic engineering and microbial engineering. The gene of a metal ABC transporter ATP binding protein ZitQ is taken as the target gene a and the lactic acid bacterium is taken as the expression host, thus successfully constructing the lactic acid bacteria which can be widely used in the preparation of food, medicine, feed and chemicals. The acid stress resistance of the engineering bacterium is significantly improved, and isimproved by 9.4 times the highest than wild strains.

Description

technical field [0001] The invention relates to a lactic acid bacteria engineering bacterium with improved acid stress resistance and an application thereof, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Lactic acid bacteria is a general term for a group of bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. These bacteria are widely distributed in nature and have rich species diversity. They are not only ideal materials for studying classification, biochemistry, genetics, molecular biology and genetic engineering, and have important academic value in theory, but also have applications in important fields closely related to human life, such as industry, agriculture and animal husbandry, food and medicine. Great value too. [0003] However, in the industrial fermentation production process of lactic acid bacteria, the problem of acid stress often exists. [0004] Acid str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12R1/01
CPCC07K14/195
Inventor 张娟杨佩珊陈坚堵国成王雅玲张淑嫣苗周迪
Owner JIANGNAN UNIV
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