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

A technology of lactic acid bacteria and engineering bacteria, applied in the field of genetic engineering and microbial engineering, can solve problems such as single method, accumulation of by-products, osmotic stress, etc., and achieve the effect of improving acid stress resistance and amino acid stress resistance

Active Publication Date: 2018-12-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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.
[0009] For amino acid stress, the current methods for enhancing strain resistance to amino acid stress such as serine mainly include: (1) adaptive evolution, which is easy to operate, but the workload is large and time-consuming; (2) timely isolation of products, Enhance the pathway of serine transport from intracellular to extracellular, and avoid the accumulation of serine in the cell. The purpose of this operation is direct, but the method is single

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of recombinant bacterial strain L lactis NZ9000 (pNZ8148 / ThiT)

[0057] Specific steps are as follows:

[0058] (1) Obtain the thiT gene sequence shown in SEQ ID NO.1 from the NCBI database, and design primers RS01735-F, RS01735-F shown in SEQ ID NO.3 and SEQ ID NO.4 respectively according to the gene sequence R;

[0059] (2) Using the genome of L. lactis NZ9000 as a template and using RS01735-F and RS01735-R as primers to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification;

[0060] (3) The PCR product and the vector pNZ8148 were double-digested with NcoI and XbaI respectively, and the digested products were purified and ligated;

[0061] (4) Transform the ligation product into Escherichia coli MC1061 (commercialized strain) competent, screen positive clones on a chloramphenicol plate, verify by colony PCR and enzyme digestion, and perform sequencing identification after the fragment size is correct, and finally obtain the co...

Embodiment 2

[0064] Embodiment 2: Growth performance test of recombinant bacterial strain L lactis NZ9000 (pNZ8148 / ThiT)

[0065] Specific steps are as follows:

[0066] (1) The bacterial strain L lactisNZ9000 (pNZ8148) (control) and the bacterial strain L lactisNZ9000 (pNZ8148 / ThiT) obtained in Example 1 were respectively inoculated in GM17 liquid medium added with 10 μg / mL chloramphenicol for activation, and placed at 30 Cultivate overnight in an incubator;

[0067] (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.;

[0068] (3) During the culturing process, samples were taken every 2 hours to measure the OD value at a wavelength of 600nm;

[0069] (4) Cultivate to OD 600 Add 10ng / mL nisin at 0.4 to induce expression of thiamine transporter, take time as abscissa, OD 600 The value is the ordinate, and the growth curve is drawn (the growth curve obtained by drawing is sh...

Embodiment 3

[0071] Example 3: Tolerance test of recombinant bacterial strain L lactis NZ9000 (pNZ8148 / ThiT) under lactic acid stress conditions

[0072] Specific steps are as follows:

[0073] The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strain L lactis NZ9000 (pNZ8148 / ThiT) obtained in Example 1 were respectively induced and cultured for 6 h, and the cells were collected by centrifugation, washed twice with 0.85% normal saline, and resuspended in an equal volume of In fresh GM17 (containing 10 μg / mL of chloramphenicol) at pH 4.0 (adjusted by lactic acid), stress for different times; wash the stressed bacterial suspension twice and resuspend in an equal volume of normal saline, take 10 μL for resuspension solution, dilute different gradient points and plant on the GM17 chloramphenicol plate to measure the number of viable bacteria and the survival rate (the results are as follows: image 3 shown).

[0074] like image 3 According to the analysis of the tol...

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Abstract

The invention discloses lactic acid bacteria engineering bacteria with improved acid stress resistance and an application thereof and belongs to the technical field of genetic engineering and microbial engineering. The lactic acid bacteria engineering bacteria capable of being widely applied to preparing food, drugs, feeds and chemicals is obtained successfully by taking a gene encoding a thiaminetransportprotein ThiT as a target gene and lactic acid bacteria as an expression host. The acid stress resistance and the amino acid stress resistance of the lactic acid bacteria engineering bacteriaare improved obviously, and the acid stress resistance and the amino acid stress resistance are improved by 12.2 and 8.4 times to the highest extent compared with those of a mild 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 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 problems of acid stress and amino acid stress often exi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/74C12R1/01
CPCC07K14/195C12N15/74
Inventor 张娟陈坚堵国成杨谨华朱政明
Owner JIANGNAN UNIV
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