Engineering lactobacillus with improved stress resistance as well as preparation method and application of engineering lactobacillus

Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a lactobacillus engineering bacterium with improved stress resistance as well as a preparation method and application thereof. In order to improve the stress resistance of lactic acid bacteria, a genetic engineering technology is utilized, and an Hpr protein for regulating and controlling an intracellular phosphotransferase system of the lactic acid bacteria is over-expressed in the lactic acid bacteria, so that the lactic acid bacteria engineering bacteria with improved stress resistance are prepared. Compared with wild type lactococcus lactis, the survival rate of the lactobacillus engineering bacterium L.lactis (ptsH) prepared by the invention under the acid stress condition that the pH value is 5.0 is increased to 43.95% from 4.49%, and the survival rate is increased by 9.79 times; under the stress of 20% NaCl (v / v), the survival rate is increased to 7.62% from 0.19%, and the survival rate is increased by 40.10 times. Therefore, when applied to industrial production and facing various stress environments, the engineering lactobacillus prepared by the invention has a huge application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genetic engineering, and specifically relates to a lactic acid bacteria engineering bacterium with improved stress resistance, a preparation method thereof, and an application thereof. Background Art

[0002] As a class of GRAS (Generally Recognized as Safe) microorganisms, lactic acid bacteria are widely used in food fermentation, biorefactories, disease treatment and other fields due to their unique metabolic characteristics and excellent industrial performance. However, when lactic acid bacteria exert their industrial functions, they inevitably encounter various stress environments, such as acid stress, oxygen stress, and salt stress, etc. These stress environments can disrupt the normal cell membrane structure of lactic acid bacteria, leading to disorders in the intracellular microenvironment and normal metabolic activities, and seriously reducing the normal industrial performance of lactic acid bacteria.

[0003] Stress environments such as acid stress, oxygen stress, and salt stress greatly reduce the efficiency of lactic acid bacteria food microbial manufacturing by seriously affecting the physiological activity of cells. Therefore, in order to improve the stress resistance of lactic acid bacteria under stress conditions, cells generally activate the intracellular stress response mechanism to resist cell damage caused by external stress environments. With the development of microbial physiology and multi-omics technologies, the intracellular response mechanism of lactic acid bacteria has been gradually revealed comprehensively. Among them, the phosphotransferase system is a class of sugar transport systems widely present in bacteria, which can couple the uptake of sugars with phosphorylation, thereby efficiently driving the transmembrane transport of sugars and directly converting them into phosphorylated forms to provide substrates for subsequent metabolism, so that lactic acid bacteria still have efficient fermentation functions under stress environments. However, the mechanism by which the phosphotransferase system enables lactic acid bacteria to have efficient fermentation functions under stress conditions remains to be further studied. Summary of the Invention

[0004] In order to improve the stress resistance of lactic acid bacteria, the present invention uses genetic engineering technology to prepare a lactic acid bacteria engineering bacterium with improved stress resistance by overexpressing the phosphotransfer protein Hpr that regulates the intracellular phosphotransferase system in lactic acid bacteria, and this protein is encoded by the gene ptsH described in the present invention.

[0005] To achieve the above application purpose, the technical solution adopted in the present application is as follows:

[0006] In the first aspect, the present invention provides a lactic acid bacteria engineering bacterium with improved stress resistance, which is obtained by introducing a recombinant plasmid containing a target gene into Lactococcus lactis to obtain a lactic acid bacteria engineering bacterium that overexpresses the Hpr protein; the target gene is the gene ptsH that regulates the intracellular phosphotransferase system of lactic acid bacteria, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] ATGGAAAAGAAAGATTTTAATGTAACTGCTGAGACAGGAATTCACGCACGTCCAGCAACTTTATTAGTACAAACTGCAAGCAAATTCAACTCTGATATTAATTTAGAGTATAAAGGTAAATCAGTAAACTTGAAATCTATTATGGGCGTTATGTCCTTAGGCGTAGGCCAAGGTTCTGATGTTACGATTTCAGTAGATGGTGCTGATGAATCAGATGCAATGGCAGCAATTGTAGAAACAATGAAAAAGGAAGGATTGTCTGAATAA。

[0009] Furthermore, the amino acid sequence of the Hpr protein is as shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:

[0011] MEKKDFNVTAETGIHARPATLLVQTASKFNSDINLEYKGKSVNLKSIMGVMSLGVGQGSDVTISVDGADESDAMAAIVETMKKEGLSE.

[0012] Furthermore, the plasmid is selected from at least one of the pNZ series, pSIP series or pLEM series. Preferably, the plasmid is pNZ8148.

[0013] Furthermore, the Lactococcus lactis is derived from Lactococcus lactis NZ9000 (L. lactis).

[0014] Furthermore, the target gene is derived from Tetragenococcus halophilus CGMCC 3792.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned engineered lactic acid bacteria, which comprises the following steps:

[0016] Recombine the gene ptsH with the plasmid, and then introduce it into Lactococcus lactis, and screen and purify it; the nucleotide sequence of the gene ptsH is as shown in SEQ ID NO.1.

[0017] Furthermore, the plasmid is selected from at least one of the pNZ series, pSIP series or pLEM series. Preferably, the plasmid is pNZ8148.

[0018] In a third aspect, the present invention provides the application of the above-mentioned engineered lactic acid bacteria in improving the stress resistance of lactic acid bacteria.

[0019] Furthermore, the stress type includes at least one of acid stress, salt stress, ethanol stress, or oxygen stress.

[0020] Beneficial effects: Since stress conditions can significantly activate the up-regulated expression of the phosphate transporter Hpr in the intracellular phosphotransferase system of lactic acid bacteria, the present invention overexpresses the phosphate transporter Hpr that regulates the intracellular phosphotransferase system in Lactococcus lactis. This protein is encoded by the gene ptsH, and the lactic acid bacteria engineering strain L. lactis (ptsH) with significantly improved stress resistance is obtained. Through experiments, it is confirmed that compared with the wild-type Lactococcus lactis, the survival rate of the lactic acid bacteria engineering strain L. lactis (ptsH) prepared in the present invention under acid stress conditions at pH = 5.0 is increased from 4.49% to 43.95%, and the survival rate is increased by 9.79 times; under 20% NaCl (v / v) stress, the survival rate is increased from 0.19% to 7.62%, and the survival rate is increased by 40.10 times. It can be seen that the lactic acid bacteria engineering strain prepared in the present invention has great application prospects when facing various stress environments, especially in the field of food fermentation. Description of the Drawings

[0021] Figure 1 Schematic diagram for the construction of the recombinant plasmid pNZ8148 / ptsH in Example 1;

[0022] Figure 2 Electrophoresis patterns of single enzyme digestion and double enzyme digestion of the recombinant plasmid pNZ8148 / ptsH in Example 1;

[0023] Figure 3 Survival rate of the recombinant strain L. lactis (ptsH) under acid stress conditions in Example 2;

[0024] Figure 4 Survival rate of the recombinant strain L. lactis (ptsH) under salt stress conditions in Example 2. Detailed Embodiments

[0025] In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present application clearer, the present application will be further described in detail below in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0026] The applicant found through experiments that stress conditions can significantly activate the up-regulated expression of the phosphotransfer protein Hpr in the phosphotransferase system within the cells of lactic acid bacteria. It is speculated that the phosphotransporter protein Hpr is a potential target for improving the stress resistance of lactic acid bacteria. Therefore, in one embodiment of the present invention, through genetic engineering and molecular biology techniques, using the Nisin-Controlled Expression System pNZ8148 as the expression backbone, the phosphotransporter gene ptsH derived from Streptococcus thermophilus was selected as the target for modification, and a recombinant plasmid was constructed by the Gibson assembly method to achieve seamless connection of the target gene and the plasmid backbone. Finally, the recombinant plasmid was electrotransformed into the competent Lactococcus lactis NZ9000 by electroporation to achieve the high-efficiency expression of the phosphotransfer protein Hpr (encoding gene ptsH) in Lactococcus lactis, thereby improving the stress resistance of lactic acid bacteria.

[0027] Among them, the most common stress environments encountered by lactic acid bacteria in conventional applications are acid stress and salt stress. Therefore, in one embodiment of the present invention, the acid stress tolerance and 20% NaCl (v / v) stress tolerance of the recombinant strain were investigated.

[0028] Specific examples will be listed below to explain the solution of the present invention. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0029] The media involved in the following examples are as follows:

[0030] Liquid LB medium: 5 g / L yeast extract powder, 10 g / L tryptone, 10 g / L sodium chloride, pH 7.0; sterilized at 121 °C for 20 min.

[0031] Solid LB medium: Liquid LB medium with an additional 2% (m / v) agar added.

[0032] Chloramphenicol was added to a final concentration of 10 μg / mL as needed.

[0033] Liquid GM17 medium: According to M17 medium (Oxoid company), with an additional supplement of glucose to 5 g / L; sterilization conditions: 121 °C, sterilized for 20 min.

[0034] Solid GM17 medium: Liquid GM17 medium with an additional 2% (m / v) agar added.

[0035] Add chloramphenicol to a final concentration of 10 μg / mL as needed.

[0036] Example 1: Construction of recombinant strain L. lactis (ptsH)

[0037] 1. Through transcriptomic analysis, it was determined that the phosphotransfer protein Hpr in the phosphotransferase system of lactic acid bacteria is related to the stress resistance of lactic acid bacteria.

[0038] 2. The nucleotide sequence of the target gene ptsH in Tetragenococcus halophilus CGMCC 3792 was obtained by whole-genome sequencing (shown in SEQ ID NO.1), and primers were designed as shown in Table 1, (Gene-F / Gene-R) to amplify the ptsH gene and purify it by gel recovery. The phosphotransfer protein gene ptsH that regulates the phosphotransferase system has a nucleotide sequence as shown in SEQ ID NO.1:

[0039] Table 1 Specific information of primers

[0040]

[0041] 3. The plasmid pNZ8148 was linearized by double digestion. The pNZ8148 was linearized using the restriction endonucleases PstI and KpnI. After double digestion, the linearized pNZ8148 fragment was purified by gel recovery.

[0042] 4. The linearized pNZ8148 fragment was ligated to the ptsH fragment using seamless cloning enzyme.

[0043] 5. The ligation product was transformed into competent Escherichia coli MC1061, spread on an LB plate containing 10 μg / mL chloramphenicol, and then placed in an incubator at 37 °C for culturing. After the formation of transformants, they were verified by colony PCR. Positive transformants were picked and inoculated into 30 mL of LB liquid medium containing 10 μg / mL chloramphenicol, cultured overnight, and then the recombinant plasmid was extracted. After verification by PCR and enzyme digestion, the recombinant plasmid pNZ8148 / ptsH( Figure 1 ) was obtained. Through electroporation technology, the recombinant plasmid was introduced into competent Lactococcus lactis NZ9000.

[0044] Among them, the electroporation conditions were as follows: 10 μL of the recombinant plasmid pNZ8148 / ptsH was mixed with 100 μL of competent Lactococcus lactis NZ9000 cells, transferred into a pre-cooled electroporation cuvette, and placed on ice for 10 min; the voltage was adjusted to 1000 V, the capacitance was 25 μF, and the resistance was 200 Ω for electrotransformation.

[0045] 6. Positive clones were screened and verified by single and double enzyme digestion( Figure 2), the correct recombinant strain L. lactis (ptsH) was obtained. Among the double digestion bands, the band at 3 kbp was the plasmid backbone, and the band at 1 kbp was pstH (about 267 bp) + egfp (about 750 bp)).

[0046] Example 2: Survival rate test of recombinant strain L. lactis (ptsH)

[0047] a. The strain L. lactis NZ9000 (control) and the strain L. lactis (ptsH) obtained in Example 1 were respectively inoculated into GM17 liquid medium containing 10 μg / mL chloramphenicol at an inoculation amount of 1%, and cultured overnight at 30 °C for activation.

[0048] b. The above-obtained seed solutions were respectively transferred to fresh GM17 liquid medium containing 10 μg / mL chloramphenicol at an inoculation amount of 2%, and cultured statically at 30 °C.

[0049] Acid stress treatment: When OD 600 = 0.2, 10 ng / mL nisin was added for inducing the expression of transport proteins. After 2 h of induction, the induced cells were taken for acid stress treatment. The stress conditions were specifically pH 5.0 and the stress time was 3 h. After the stress ended, gradient dilution and plate counting were performed to calculate the survival rates of the recombinant strain and the control strain under acid stress.

[0050] The results were as Figure 3 shown. The survival rate of the recombinant strain under acid stress conditions was higher than that of the control strain. The survival rate increased from 4.49% to 43.95%, and the survival rate increased by 9.79 times.

[0051] 20% NaCl stress treatment was as follows: When OD 600 = 0.2, 10 ng / mL nisin was added for inducing the expression of transport proteins. After 2 h of induction, the induced cells were taken for salt stress treatment. The stress conditions were specifically 20% NaCl (v / v) and the stress time was 3 h. After the stress ended, gradient dilution and plate counting were performed to calculate the survival rates of the recombinant strain and the control strain under salt stress.

[0052] The results were as Figure 4 shown. The survival rate of the recombinant strain under salt stress conditions was higher than that of the control strain. The survival rate increased from 0.19% to 7.62%, and the survival rate increased by 40.10 times.

[0053] Thus, it can be seen that by overexpressing the gene ptsH (phosphotransferase Hpr) that regulates the intracellular phosphotransferase system of lactic acid bacteria, the present invention can improve the acid stress resistance of lactic acid bacteria.

Claims

1. Engineered lactic acid bacteria with improved stress resistance, characterized in that: Recombinant plasmid containing the target gene is introduced into Lactococcus lactis to obtain an engineered lactic acid bacterium strain that overexpresses the Hpr protein; the target gene is the gene ptsH that regulates the phosphotransferase system in lactic acid bacteria, and its nucleotide sequence is as shown in SEQ ID NO.

1.

2. The engineered lactic acid bacteria with enhanced stress resistance according to claim 1, wherein: The amino acid sequence of the Hpr protein is as shown in SEQ ID NO.

2.

3. The engineered lactic acid bacteria with improved stress resistance according to claim 1, characterized in that: The plasmid is selected from at least one of the pNZ series, pSIP series or pLEM series.

4. The engineered lactic acid bacteria with improved stress resistance according to claim 1, characterized in that: The Lactococcus lactis is derived from Lactococcus lactis NZ9000.

5. The engineered lactic acid bacteria strain with improved stress resistance according to claim 1, characterized in that: The target gene is derived from Tetragenococcus halophilus CGMCC 3792.

6. The preparation method of the engineered lactic acid bacteria according to any one of claims 1 to 5, characterized in that: It includes the following steps: Recombine the gene ptsH with the plasmid, and then introduce it into Lactococcus lactis, and screen and purify it; the nucleotide sequence of the gene ptsH is as shown in SEQ ID NO.

1.

7. The preparation method of the genetically engineered lactic acid bacteria according to claim 6, characterized in that: The plasmid is selected from at least one of the pNZ series, pSIP series or pLEM series.

8. Use of the engineered lactic acid bacterium strain according to any one of claims 1 to 5 in improving the stress resistance of lactic acid bacteria.

9. The application according to claim 8, wherein: The stress type includes at least one of acid stress, salt stress, ethanol stress or oxygen stress.

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