Lactococcus lactis acid-resistant gene module expression plasmid as well as construction method and application thereof

Abstract

The invention discloses a lactococcus lactis acid-resistant gene module expression plasmid as well as a construction method and application thereof. The expression plasmid of the acid-resistant gene module of the lactococcus lactis comprises a KGA92937 leptospirillum ferriphilum chaperonin gene and a pNZ8148 plasmid skeleton. The construction method comprises the following steps: artificially synthesizing a KGA92937 leptospirillum ferriphilum chaperonin gene segment, and carrying out PCR (Polymerase Chain Reaction) amplification on a pNZ8148 linear plasmid skeleton; the two gene segments are assembled through a seamless cloning technology and chemically converted to enter MC1061 Escherichia coli competent cells; and screening correct clones by using primers, extracting plasmids, sequencing and identifying to obtain the expression plasmids of the acid-resistant gene module of the lactococcus lactis. According to the acid-resistant gene module expression plasmid obtained by the method, the tolerance of lactococcus lactis cells under an extreme acidic condition can be remarkably improved, and the acid stress survival rate of an acid-resistant module gene transformant is improved by 76% compared with that of an empty plasmid control.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to an acid-tolerant gene module expression plasmid of Lactococcus lactis, a construction method thereof, and applications thereof. Background Art

[0002] Lactococcus lactis is a spherical facultative anaerobic Gram-positive bacterium, which has been widely used in the fermentation of foods such as cheese, yogurt, and pickles; it is also the main producing strain of nisin. Nisin can be used as a green food preservative without toxic side effects and has been widely used in meats, dairy products, etc. around the world. As a safe model strain, Lactococcus lactis can be used as a microbial cell factory through the application of biotechnology and genetic engineering to produce recombinant proteins and metabolites. During the industrial production fermentation process, lactic acid bacteria secrete lactic acid due to energy metabolism, and the accumulation of lactic acid will lower the pH of the fermentation broth to about 4.2, thereby inhibiting the growth of bacteria and reducing the sugar decomposition rate, and further affecting the survival of the bacteria and the production of nisin.

[0003] Extremophiles refer to microorganisms that live in extreme environments, such as high temperature, high pressure, high salt, low temperature and other extreme environments. These microorganisms have strong adaptability and survival ability. They can utilize the energy and compounds in the extreme environment to maintain their own survival and reproduction, and are widely regarded as an important innovation direction for the next generation of industrial biotechnology. Extreme acidophilic microorganisms are a class of microorganisms that can survive in extreme acidic environments, with an optimal growth pH value below 3.0, and are isolated from extreme acidic environments such as acid mine drainage and geothermal springs. Acidophilic microorganisms have important application values in the fields of bioleaching, acid wastewater treatment, biohydrometallurgy, microbial fuel cells, and food and medicine. The enzymes derived from heat-resistant and acid-resistant bacteria extracted from acidophilic microorganisms have high activities at low pH and high temperature, which have great potential value in industrial applications such as starch, juice, feed, and baking.

[0004] Leptospirillum ferriphilum (L. ferriphilum) lives in an environment with extremely low pH (pH 1.5 - 1.8), and its optimal growth temperature (40°C - 45°C) is higher than that of most extremely acidophilic microorganisms. Therefore, exploring the acid-resistant functional elements of Leptospirillum ferriphilum strains can, to a certain extent, solve the problem of the lack of functional modules in extremely acidophilic microorganisms. Chaperone proteins in cells can bind to other proteins to form complexes and cooperate to complete the repair and protection of biological macromolecules. During the repair of DNA damaged by an acidic environment, chaperone proteins can bind to DNA damage recognition proteins and cooperate to complete the repair of DNA damage. It is a typical acid-resistant gene and has the effect of enhancing the acid tolerance of cells. Summary of the Invention

[0005] To overcome the deficiency in the prior art that lactic acid bacteria secrete lactic acid due to energy metabolism, and the accumulation of lactic acid will lower the pH of the fermentation broth to about 4.2, thereby inhibiting the growth of bacteria and reducing the sugar decomposition rate, the main object of the present invention is to provide a plasmid for expressing an acid-resistant gene module of Lactococcus lactis, its construction method and application. It solves the problem of the low survival rate of bacteria in an acidic fermentation broth.

[0006] To achieve the aforementioned invention object, the technical solutions adopted by the present invention include:

[0007] A plasmid for expressing an acid-resistant gene module of Lactococcus lactis, comprising the chaperone protein gene of Leptospirillum ferriphilum KGA92937 and the plasmid backbone of pNZ8148; the plasmid for expressing an acid-resistant gene module of Lactococcus lactis is named pNZ8148-KGA92937, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The nucleotide sequence of the chaperone protein gene of Leptospirillum ferriphilum KGA92937 is shown in SEQ ID NO.2; the nucleotide sequence of the linear plasmid backbone of pNZ8148 is shown in SEQ ID NO.3.

[0009] The plasmid backbone of pNZ8148 contains the PnisA inducible promoter and a terminator.

[0010] A method for constructing a plasmid for expressing an acid-resistant gene module of Lactococcus lactis, comprising the following steps:

[0011] 1) Artificially synthesize the chaperone protein gene fragment of Leptospirillum ferriphilum KGA92937, and PCR amplify the linear plasmid backbone of pNZ8148;

[0012] 2) Assemble the two gene fragments in step 1) by seamless cloning technology and chemically transform them into competent cells of Escherichia coli MC1061;

[0013] 3) Use primers to screen for correct clones, extract and sequence the plasmids for identification, and obtain the expression plasmid pNZ8148-KGA92937 of the acid tolerance gene module of Lactococcus lactis.

[0014] In step 1), the KGA92937 gene of Leptospira interrogans ferritin-binding protein is obtained by PCR amplification using primers K937-F and K937-R. The nucleotide sequence of primer K937-F is as shown in SEQ ID NO.6, and the nucleotide sequence of primer K937-R is as shown in SEQ ID NO.7.

[0015] In step 1), the linear plasmid backbone of pNZ8148 is obtained by PCR amplification using plasmid pNZ8148 as a template and primers L8148-F and PniZ20-R. The nucleotide sequence of primer L8148-F is as shown in SEQ ID NO.8, and the nucleotide sequence of primer PniZ20-R is as shown in SEQ ID NO.9.

[0016] Application of the expression plasmid of the acid tolerance gene module of Lactococcus lactis in improving acid tolerance in Lactococcus lactis.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention provides a plasmid with Lactococcus lactis as the host, which carries and expresses the gene module of Leptospira interrogans ferritin-binding protein, and can significantly improve the tolerance of Lactococcus lactis cells under extreme acidic conditions. The acid stress survival rate of the acid tolerance module gene transformant is increased by 76% compared with the empty plasmid control. Brief Description of the Drawings

[0019] Figure 1 : Schematic diagram of the structure of the expression plasmid pNZ8148-KGA92937 of the acid tolerance gene module of Lactococcus lactis.

[0020] Figure 2 : Comparison chart of the acid stress survival rates of pNZ8148-KGA92937 transformants and the empty plasmid control. Detailed Embodiments

[0021] The present invention will be further described below in conjunction with specific embodiments. These embodiments are exemplary only and do not limit the protection scope of the present invention. Lactococcus lactis NZ9000 and Escherichia coli MC1061 involved in each embodiment can be obtained by purchase.

[0022] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0023] Example 1 Construction of an Acid-Resistant Gene Module Expression Plasmid for Lactococcus lactis

[0024] Step 1. The codons of the DNA chaperone protein sequence of Leptospira ferrooxidans KGA92937 in the National Center for Biotechnology Information (NCBI) database were optimized. According to the obtained gene sequence, the DNA chaperone protein gene of Leptospira ferrooxidans KGA92937 was artificially synthesized by a company and amplified with primers K937-F (SEQ ID NO.6) and K937-R (SEQ ID NO.7) to obtain a Leptospira ferrooxidans chaperone protein gene fragment (SEQ ID NO.2). Using plasmid pNZ8148 as the backbone, it was amplified with primers L8148-F (SEQ ID NO.8) and PniZ20-R (SEQ ID NO.9) to obtain a linear plasmid backbone fragment of pNZ8148 (SEQ ID NO.3), which contains the PnisA inducible promoter (SEQ ID NO.4) and the terminator sequence (SEQ ID NO.5).

[0025] Step 2. The two gene fragments, namely the Leptospira ferrooxidans chaperone protein gene fragment (SEQ ID NO.2) and the linear plasmid backbone fragment of pNZ8148 (SEQ ID NO.3), were assembled by seamless cloning using the Xinsaimi Seamless Cloning Kit (M9001). The molar ratio of the added fragments was 1:1, and the structure is as Figure 1 shown.

[0026] Step 3. Transformation of the assembled product into competent cells of Escherichia coli MC1061: Add 10 μL of the above-mentioned assembly reaction solution into 50 μL of thawed commercial competent cells of Escherichia coli MC1061. After ice-bathing for 30 min, perform heat shock in a 42 °C water bath for 60 s, and then quickly transfer it to an ice bath for 2 min. Add 500 μL of LB medium, and culture it in a shaking incubator at 220 r / min and 37 °C for 45 min. Centrifuge to discard 400 μL of the supernatant, mix the remaining bacterial solution, and spread it on an LB solid plate containing chloramphenicol (final concentration: 25 μg / mL), and culture it overnight at 37 °C. Use primer K937-F (SEQ ID NO.6) and primer K937-R (SEQ ID NO.7) for colony PCR verification. The correct cloned band length is 1676 bp. Culture the clones with the correct band using LB containing chloramphenicol resistance, extract the plasmid using Tiangen's plasmid mini-prep kit (DP103), and send it to the company for sequencing. Name the correct clone pNZ8148-KGA92937 (SEQ ID NO.1).

[0027] Example 2. Transformation of the acid tolerance gene module expression plasmid of Lactococcus lactis into Lactococcus lactis NZ9000

[0028] Step 1. Preparation of electrocompetent cells of Lactococcus lactis

[0029] Take the glycerol bacteria of Lactococcus lactis L.lactic NZ9000 stored frozen in the laboratory, perform three-zone streaking on a GM17 agar medium plate, and then place it in a 30 °C incubator for static culture. The next day, inoculate the grown single colonies into GM17 liquid medium, passage and activate them three times, and then inoculate them into 50 mL of GM17 broth, and statically culture at 30 °C for about 3 - 4 h until the logarithmic growth phase (OD600 ≈ 0.5). Add ampicillin with a final concentration of 20 μg / mL, and continue to statically culture at 30 °C for 1 h to weaken the cell wall. Use two 50 mL centrifuge tubes to collect the bacterial cell precipitate. Resuspend the bacterial cell precipitate with 20 mL of 10% glycerol, centrifuge at 8000 rmp for 3 min, repeat this step 3 times, and then resuspend the bacterial cell precipitate with 1 mL of 10% glycerol. Aliquot 50 μL per tube into 1.5 mL EP tubes, and quickly place them in an -80 °C refrigerator for storage and standby.

[0030] Step 2. Electrotransformation of Lactococcus lactis

[0031] Take the electrocompetent cells of L. lactic NZ9000 stored at -80°C and thaw them on ice. Pipette 500 ng of the empty plasmid pNZ8148 and 500 ng of pNZ8148-KGA92937 into 50 μL of the electrocompetent cells of L. lactic NZ9000 respectively. After gently pipetting and mixing, transfer the mixture to a pre-cooled 2 mm electroporation cuvette. Set the voltage of the electroporator to 2.5 kV, press the "pulse" button to initiate the electroporation, and then quickly add 1 mL of GM17 broth for recovery. After mixing, transfer the mixture to a 1.5 mL sterile EP tube and incubate at 30°C for 2 h. After centrifuging at 8000 rpm for 2 min, discard 900 μL of the supernatant, spread the cells on a GM17 plate containing chloramphenicol (10 μg / mL) and culture at 30°C for 16 h. When obvious colonies appear on the plate, pick the colonies and culture and amplify them in GM17 liquid containing chloramphenicol (10 μg / mL).

[0032] Example 3 Acid Stress Experiment of Lactococcus lactis Transformants

[0033] The Lactococcus lactis pNZ8148-KGA92937 transformants and the control pNZ8148 empty plasmid transformants were respectively subcultured and activated in GM17 liquid medium containing chloramphenicol (10 μg / mL) (inoculum size 2%). When subculturing for the third time, culture them in GM17 liquid medium containing 25 ng / mL Nisin and chloramphenicol (10 μg / mL). Take 1 mL of the culture broth that has grown to the late logarithmic phase (about 7 h). Among them, take 100 μL and quickly dilute it to 10-6 in sterile physiological saline. Pipette 100 μL and spread it on a GM17 plate containing chloramphenicol (10 μg / mL) and culture at 30°C for 16 h. Set 3 parallels. The remaining 900 μL of the late logarithmic phase culture broth was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, an equal volume of GM17 liquid medium with pH 3.0 was added for acid stress for 3 h, centrifuged at 8000 rmp for 3 min, the supernatant was discarded, and the cells were resuspended with an equal volume of physiological saline. Take 100 μL and quickly dilute it to 10 -6 , pipette 100 μL and spread it on a GM17 plate containing chloramphenicol (10 μg / mL) and culture at 30°C for 16 h. Set 3 parallels. Count the number of colonies on each plate, and divide the number of colonies after treatment with the GM17 liquid medium at pH 3.0 by the number of colonies before acid stress to calculate the survival rate of the pNZ8148-KGA92937 transformants and the pNZ8148 empty plasmid transformants after acid stress treatment.

[0034] Survival rate = Number of viable cells (cfu) before acid stress / Number of viable cells (cfu) after acid stress

[0035] The results are as follows Figure 2As shown, the acid stress survival rate of the pNZ8148-KGA92937 transformant was increased by 76% compared to the empty plasmid control.

[0036] The above-mentioned embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Claims

1. An acid-resistant gene module expression plasmid of Lactococcus lactis, characterized in that, It contains the chaperone protein gene of Leptospira ferrooxidans KGA92937 and the plasmid backbone of pNZ8148; the acid-tolerant gene module expression plasmid of Lactococcus lactis is named pNZ8148-KGA92937, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The Lactococcus lactis acid-resistant gene module expression plasmid according to claim 1, characterized in that, The nucleotide sequence of the chaperone protein gene of Leptospira ferrooxidans KGA92937 is shown in SEQ ID NO.2; the nucleotide sequence of the linear plasmid backbone of pNZ8148 is shown in SEQ ID NO.

3.

3. The Lactococcus lactis acid-resistant gene module expression plasmid according to claim 1, characterized in that, The plasmid backbone of pNZ8148 contains the PnisA inducible promoter and terminator.

4. A method for constructing an expression plasmid of an acid-resistant gene module of Lactococcus lactis, characterized in that, It includes the following steps: 1) Artificially synthesize the chaperone protein gene fragment of Leptospira ferrooxidans KGA92937, and PCR amplify the linear plasmid backbone of pNZ8148; 2) Assemble the two gene fragments in step 1) by seamless cloning technology and chemically transform them into competent cells of Escherichia coli MC1061; 3) Use primers to screen for correct clones, extract plasmids and identify them by sequencing to obtain the acid-tolerant gene module expression plasmid pNZ8148-KGA92937 of Lactococcus lactis.

5. The method for constructing a plasmid for expressing an acid-resistant gene module of Lactococcus lactis according to claim 4, characterized in that, In step 1), the chaperone protein gene of Leptospira ferrooxidans KGA92937 is obtained by PCR amplification using primer K937-F and primer K937-R. The nucleotide sequence of primer K937-F is shown in SEQ ID NO.6, and the nucleotide sequence of primer K937-R is shown in SEQ ID NO.

7.

6. The method for constructing a plasmid for expressing an acid-resistant gene module of Lactococcus lactis according to claim 4, characterized in that In step 1), the linear plasmid backbone of pNZ8148 is obtained by PCR amplification using plasmid pNZ8148 as a template and primers L8148-F and PniZ20-R. The nucleotide sequence of primer L8148-F is shown in SEQ ID NO.8, and the nucleotide sequence of primer PniZ20-R is shown in SEQ ID NO.

9.

7. Application of the acid-tolerant gene module expression plasmid of Lactococcus lactis described in claim 1 in enhancing acid tolerance in Lactococcus lactis.

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