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Lactobacillus engineered bacterium with improved acid stress resistance and application thereof

A technology of lactic acid bacteria and engineering bacteria, which is applied in the field of genetic engineering and microbial engineering, can solve the problems of by-product accumulation, osmotic stress, and low success rate, and achieve the effect of improving acid stress resistance

Active Publication Date: 2019-03-19
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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  • Lactobacillus engineered bacterium with improved acid stress resistance and application thereof
  • Lactobacillus engineered bacterium with improved acid stress resistance and application thereof
  • Lactobacillus engineered bacterium with improved acid stress resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: Construction of recombinant bacterial strain

[0062] Specific steps are as follows:

[0063] (1) Obtain the ecfA2 gene sequence shown in SEQ ID NO.1 from the NCBI database (the gene encoding the energy coupling factor transporter ATPase EcfA2, the protein used to transport ATP on the membrane), such as SEQ ID NO. The zitP gene sequence shown in 3 (the gene encoding the metal ion ABC transporter permease ZitP, the protein used to transport metal ions on the membrane), the zitQ gene sequence as shown in SEQ ID NO.4 (encoding the metal ion ABC transporter The gene of the ATP-binding protein ZitQ, the protein used to transport metal ions on the membrane), the bglF gene sequence shown in SEQ ID NO.5 (the gene encoding the IIABC component BglF of the β-glucoside specific PTS system, used The protein used to transport β-glucoside), the ganP gene sequence shown in SEQ ID NO.6 (the gene encoding maltose ABC transporter permease GanP, the protein used to transport...

Embodiment 2

[0071] Embodiment 2: the growth performance test of recombinant bacterial strain

[0072] Specific steps are as follows:

[0073] (1) The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strain L lactis NZ9000 (pNZ8148 / ecfA2), L lactis NZ9000 (pNZ8148 / zitP), L lactis NZ9000 (pNZ8148 / zitQ), Llactis NZ9000 ( pNZ8148 / bglF) and 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;

[0074] (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.;

[0075] (3) During the culturing process, samples were taken at regular intervals to measure the OD value at a wavelength of 600nm;

[0076] (4) Cultivate to OD 600 Add 10ng / mL nisin at 0.4 to induce the expression of the transporter, take time as the abscissa...

Embodiment 3

[0080] Example 3: Tolerance test of recombinant strains under lactic acid stress conditions

[0081] Specific steps are as follows:

[0082] The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strains L lactis NZ9000 (pNZ8148 / ecfA2), L lactis NZ9000 (pNZ8148 / zitP) and lactis NZ9000 (pNZ8148 / zitQ) obtained in Example 1 were induced and cultured for 6 h, and the cells were collected by centrifugation , washed twice with 0.85% normal saline, resuspended in an equal volume of fresh GM17 (containing chloramphenicol 10 μg / mL) at pH 4.0 (adjusted by lactic acid), and stressed for different times; the bacterial suspension after stress was washed Resuspend in equal volume of normal saline after two times, take 10 μL of resuspension, dilute different gradients and plant on GM17 chloramphenicol plate to determine the number of viable bacteria and survival rate (the results are as follows: Figure 9-11 shown);

[0083] Survival rate = (N / N 0 )×100%;

[0084] Amo...

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Abstract

The invention discloses a Lactobacillus engineered bacterium with improved acid stress resistance and application thereof, and belongs to the technical field of genetic engineering and microbial engineering. With a gene of an encoded energy-coupling factor transporter ATP enzyme cfA2 serving as a target gene and Lactobacillus as an expression host, the Lactobacillus engineered bacterium widely applicable to the preparation of foods, drugs, feeds and chemicals is successfully constructed; acid stress resistance of the Lactobacillus engineered bacterium is evidently improved, which is 598.7 times as high as that of a wild strain.

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 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 production of lactic acid bacteria and their function as probiotics in the human gastrointesti...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12R1/01
CPCC12N9/14C12N15/746C12Y306/04008
Inventor 张娟杨佩珊刘为佳陈坚堵国成
Owner JIANGNAN UNIV
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