CN110846339A - Method for improving acid stress resistance of serratia marcescens - Google Patents
Method for improving acid stress resistance of serratia marcescens Download PDFInfo
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Abstract
The invention discloses a method for improving acid stress resistance of serratia marcescens, and belongs to the technical field of genetic engineering and microbial engineering. The acid stress resistance of the serratia marcescens is obviously improved by over-expressing the DNA binding protein XrpA in the serratia marcescens; the survival rate of the recombinant serratia marcescens prepared by the method of the invention after being cultured for 12 hours in the environment with pH of 4.0 is 1.4 times of that of wild serratia marcescens.
Description
Technical Field
The invention relates to a method for improving acid stress resistance of serratia marcescens, belonging to the technical field of genetic engineering and microbial engineering.
Background
Serratia marcescens (Serratia marcocens) is an important industrial strain, is often used for producing prodigiosin, 2, 3-butanediol and other fermentation products which are widely applied to the preparation of foods, medicines and daily chemical products, and has wide market prospect.
However, in the fermentation production process of Serratia marcescens (Serratia marcocens), the fermentation production process inevitably faces various environmental stresses from external environment, such as acid stress, ethanol stress, oxygen stress, salt stress and the like, and the environmental stresses all seriously affect the physiological activity of the Serratia marcescens (Serratia marcocens), thereby seriously limiting the fermentation performance of the Serratia marcescens (Serratia marcocens).
Among the numerous environmental stresses faced by Serratia marcescens (Serratia marcescens), acid stress is the most severe. Acid stress is caused by accumulation of organic acids such as lactic acid and acetic acid secreted to the outside of cells by Serratia marcescens (Serratia marcocens), and because the pH value in the cells is usually higher than that in the cells, the accumulated acidic substances such as the lactic acid and the acetic acid can enter the cells through passive diffusion, and the organic acids such as the lactic acid and the acetic acid entering the cells are rapidly dissociated, so that the pH value in the cells is rapidly reduced, and the cells are subjected to severe acid stress.
In order to maintain the fermentation performance of Serratia marcescens (Serratia marcescens), it has been common in the past to add CaCO during fermentation of Serratia marcescens (Serratia marcescens)3And an alkaline substance such as NaOH to maintain the pH in a stable range.
However, the addition of alkaline substances often causes the accumulation of byproducts in the fermentation process of Serratia marcescens (Serratia marcescens), and salts formed by the byproducts can cause the cells to be in a hypertonic environment again, so that osmotic stress is generated, and the growth and metabolism of the Serratia marcescens (Serratia marcescens) are influenced again. Therefore, there is an urgent need to find a method for improving the acid stress resistance of lactic acid bacteria with better effect.
Disclosure of Invention
[ problem ] to
The invention aims to provide a method for improving the acid stress resistance of serratia marcescens.
[ solution ]
In order to solve the technical problems, the invention provides a method for improving the acid stress resistance of Serratia marcescens, which comprises the steps of over-expressing DNA binding protein XrpA in the Serratia marcescens; the DNA binding protein XrpA is a protein on the cell membrane of microorganisms for recognizing and binding DNA.
In one embodiment of the invention, the amino acid sequence of the DNA binding protein XrpA is shown in SEQ ID NO. 1.
In one embodiment of the invention, the nucleotide sequence of the gene encoding the DNA binding protein XrpA is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the overexpression is performed by ligating the gene encoding the DNA binding protein XrpA with an expression vector to construct a recombinant plasmid containing the gene encoding the DNA binding protein XrpA, and then introducing the recombinant plasmid into Serratia marcescens.
In one embodiment of the invention, the expression vector is a pET-28a (+) plasmid.
In one embodiment of the invention, the overexpression is to perform double enzyme digestion on the gene coding the DNA binding protein XrpA and the pET-28a (+) plasmid by Ecor I and BamH I to obtain an enzyme digestion product, then link the enzyme digestion product to construct a recombinant plasmid containing the gene coding the DNA binding protein XrpA, and finally introduce the recombinant plasmid into Serratia marcescens
The invention also provides a recombinant serratia marcescens which is prepared by the method for improving the acid stress resistance of the serratia marcescens.
The invention also provides a method for producing prodigiosin, which comprises the steps of inoculating the recombinant serratia marcescens into a fermentation culture medium for culture to obtain fermentation liquor, and extracting prodigiosin from the fermentation liquor.
The invention also provides a method for producing 2, 3-butanediol, which is characterized in that the method comprises the steps of inoculating the recombinant serratia marcescens into a fermentation culture medium for culture to obtain a fermentation broth, and extracting the 2, 3-butanediol from the fermentation broth.
The invention also provides the application of the method for improving the acid stress resistance of the serratia marcescens, the recombinant serratia marcescens, the method for producing prodigiosin or the method for producing 2, 3-butanediol in preparing foods, medicines and daily chemical products.
[ advantageous effects ]
(1) The acid stress resistance of the serratia marcescens is obviously improved by over-expressing the DNA binding protein XrpA in the serratia marcescens; the survival rate of the recombinant serratia marcescens prepared by the method of the invention after being cultured for 12 hours in the environment with pH of 4.0 is 1.4 times of that of wild serratia marcescens.
(2) According to the invention, the recombinant serratia marcescens with remarkably improved acid stress resistance is obtained by over-expressing the DNA binding protein XrpA in the serratia marcescens; the survival rate of the recombinant serratia marcescens of the invention after being cultured for 12h under the environment with pH of 4.0 is 1.4 times of that of wild serratia marcescens.
(3) The method for improving the acid stress resistance of the serratia marcescens has good effect, does not cause any influence on the growth performance of the serratia marcescens, and is suitable for large-scale industrial production.
Drawings
FIG. 1: plasmid map of recombinant plasmid pET-28 a-xrpA.
FIG. 2: growth curves of recombinant Serratia marcescens/pET-28a-Vector (control), recombinant Serratia marcescens/pET-28a-xrpA and recombinant Serratia marcescens/pET-28 a-xrpA.
FIG. 3: the growth of cells of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA at pH 4.
FIG. 4: the growth of cells of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA at pH 5.
FIG. 5: the growth of cells of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA at pH 6.
FIG. 6: the growth of cells of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA at pH 7.
FIG. 7: growth curves of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA at pH 4.
Detailed Description
The invention is further illustrated with reference to specific examples.
The pET-28a (+) plasmid referred to in the examples below was purchased from Novagen; serratia marcescens (Serratia marcocens) referred to in the examples below was obtained from North Navy organism under the product number BNCC 336646.
The media involved in the following examples are as follows:
LB liquid medium: 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride.
LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 20g/L agar.
Example 1: construction of recombinant strains
The method comprises the following specific steps:
(1) chemically synthesizing an xrpA gene having a nucleotide sequence shown in SEQ ID No.2 (the xrpA gene is a gene encoding a DNA binding protein xrpA, the DNA binding protein xrpA is a protein on a microbial cell membrane for recognizing and binding to DNA, belonging to XRE family), an xrpB gene having a nucleotide sequence shown in SEQ ID No.3 (the xrpB gene is a gene encoding xrpB, the xrpB is a protein on a microbial cell membrane for recognizing and binding to DNA, belonging to XRE family), an xrpC gene having a nucleotide sequence shown in SEQ ID No.4 (the xrpC gene is a gene encoding xrpC, the xrpC is a protein on a microbial cell membrane for recognizing and binding to DNA, belonging to XRE family), an xrpD gene having a nucleotide sequence shown in SEQ ID No.5 (the xrpD gene is a gene encoding xrpD, the xrd is a protein on a microbial cell membrane for recognizing and binding to DNA, belonging to LysR family);
(2) carrying out double enzyme digestion on the synthesized gene and the pET-28a (+) plasmid respectively by using the restriction enzyme in the table 1, and connecting after purifying enzyme digestion products; introducing the obtained ligation product into Serratia marcescens (Serratiamarcescens); the transformed Serratia marcescens (Serratia marcescens) is streaked on LB solid medium containing 50 mug/mL kanamycin and cultured for 16h at 30 ℃; selecting positive transformants to be inoculated in LB liquid culture medium containing 50 mug/mL kanamycin, culturing for 12h at 30 ℃ and 180rpm, collecting thalli, extracting plasmids, carrying out electrophoresis verification after the plasmids are subjected to double enzyme digestion by restriction enzymes in Table 1, and carrying out sequencing verification on the plasmids to verify the plasmids to obtain the recombinant Serratia marcescens/pET-28a-xrpA (the plasmid map of the recombinant plasmid pET-28a-xrpA is shown in figure 1), the recombinant Serratia marcescens/pET-28a-xrpA, and the recombinant Serratia marcescens/pET-28 a-xPD.
TABLE 1 restriction enzymes used for the different genes
| Gene | Restriction enzyme |
| xrpA | Ecor I、BamH I |
| xrpB | Ecor I、BamH I |
| xrpC | Ecor I、BamH I |
| xrpD | Ecor I、BamH I |
Example 2: growth Performance test of recombinant strains
The method comprises the following specific steps:
respectively scribing recombinant Serratia marcescens Serratia marcocens/pET-28 a-Vector (control) only containing blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcocens/pET-28 a-xrpA, the recombinant Serratia marcescens Serratia marcocens/pET-28 a-xrpA C and the recombinant Serratia marcescens Serratia marcocens/pET-28 a-xrPD on LB solid culture medium, and culturing for 16h at 30 ℃; selecting positive transformants, inoculating the positive transformants into an LB liquid culture medium containing 50 mu g/mL kanamycin, and culturing for 12h at 30 ℃ and 180rpm to obtain a seed solution; respectively inoculating the seed liquid into LB liquid culture medium, controlling initial OD600The culture was carried out at 30 ℃ and 180rpm (0.1), and the OD of the culture solution was measured by sampling the culture solution every two hours600Values, growth curves were plotted (see fig. 2).
The results are shown in FIG. 2, and the analysis of growth performance tests shows that the biomass of the recombinant Serratia marcescens/pET-28a-xrpA and the recombinant Serratia marcescens/pET-28a-xrpC have no great difference from the control strain, which indicates that the overexpression of XrpA and XrpC proteins in Serratia marcescens has no influence on the growth performance of the strain, while the biomass of the recombinant Serratia marcescens/pET-28a-xrpB and the recombinant Serratia marcescens/pET-28a-xrpD is obviously lower than that of the control strain, which indicates that the overexpression of XrpB and XrpD proteins in Serratia marcescens can inhibit the growth of the strain.
Example 3: tolerance test of recombinant strains under acid stress conditions
The method comprises the following specific steps:
respectively scribing recombinant Serratia marcescens Serratia marcescens/pET-28a-Vector (control) only containing blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA and the recombinant Serratia marcescens/pET-28a-xrpC obtained in example 1 on LB solid culture medium, and culturing for 16h at 30 ℃ to obtain single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 12h under the conditions of 30 ℃ and 180rpm to obtain seed liquid; respectively inoculating the seed liquid into LB liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 30 deg.C and 180rpm for 12h to obtain culture solution; collecting part of culture solution, centrifugally collecting cells, washing the collected cells twice by 0.85% physiological saline, then suspending the cells in LB liquid culture medium with pH of 4.0 (adjusted by HCl) and equal volume to the collected culture solution, and respectively stressing for 12h to obtain bacterial suspension; collecting partial bacterial suspension, centrifugally collecting cells, washing the collected cells twice by 0.85% physiological saline, and then suspending the cells in physiological saline with the same volume as the collected bacterial suspension to obtain bacterial liquid; 200 μ L of the resuspension solution was plated on LB solid medium containing 50 μ g/mL kanamycin to determine viable cell count and viability (results are shown in Table 2);
wherein the survival rate is (N/N)0)×100％；
In the formula, N0Is the number of viable colonies of the bacterial suspension without acid stress treatment on LB solid culture medium containing 50 mug/mL kanamycin; n is the number of viable colonies grown on LB solid medium containing 50. mu.g/mL kanamycin after 12h of stress.
As can be seen from Table 2, through tolerance experiment analysis, after 12 hours of stress in LB liquid medium with pH 4.0, the survival rate of the recombinant Serratia marcescens/pET-28a-xrpA is 1.4 times of that of the control, which shows that the tolerance of the recombinant Serratia marcescens/pET-28a-xrpA to acid stress is obviously improved; after 12 hours of stress in LB liquid culture medium with pH 4.0, the survival rate of the recombinant Serratia marcescens/pET-28a-xrPC is only 0.9 times of that of the control, which shows that the tolerance of the recombinant Serratia marcescens/pET-28a-xrPC to acid stress is not improved. This demonstrates that the DNA binding protein XrpA is able to regulate the cell's tolerance to osmotic stress, whereas other proteins are unable.
TABLE 2 Effect of acid stress on survival of different recombinant strains
Example 4: growth performance test of recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA under acid stress condition
The method comprises the following specific steps:
1. plate experiment
The recombinant Serratia marcescens Serratia marcescens/pET-28a-Vector (comparison) only containing the blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA obtained in the example 1 are respectively streaked on an LB solid culture medium and cultured for 16h at the temperature of 30 ℃ to obtain a single colony; selecting single colonies, respectively inoculating to LB liquid culture medium, and culturing at 30 deg.C and 180rpm for 12 hr to obtain first-stage seed solution; respectively inoculating the seed liquid into an LB liquid culture medium, and culturing for 2h to logarithmic phase at 30 ℃ and 180rpm to obtain a secondary seed liquid; determining thallus concentration of the second-stage seed liquid and determining OD of the second-stage seed liquid600Adjusting the value to 0.8, taking the initial concentration as the value, and carrying out 10-time gradient dilution on OD600 for 6 times to obtain a bacterial liquid; respectively dibbling 1 μ L of the bacterial solution on LB solid culture medium with pH of 4, 5, 6 and 7, and culturing at 30 deg.C for 24 h; 24hThereafter, the growth of the cells was observed and photographed (see FIGS. 3 to 6).
2. Growth curve
The recombinant Serratia marcescens Serratia marcescens/pET-28a-Vector (comparison) only containing the blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA obtained in the example 1 are respectively streaked on an LB solid culture medium and cultured for 16h at the temperature of 30 ℃ to obtain a single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 12h under the conditions of 30 ℃ and 180rpm to obtain seed liquid; inoculating the seed solution into LB liquid culture medium with pH of 4, and controlling initial OD600The culture was carried out at 30 ℃ and 180rpm (0.1), and the OD in the culture solution was measured by sampling every two hours600Values, growth curves were plotted (see fig. 7 for growth curves).
3-7, the growth performance of the recombinant Serratia marcescens/pET-28a-xrpA is better than that of the control group under the acid stress condition, which further proves that the DNA binding protein XrpA can regulate the tolerance of the cell to osmotic stress.
Example 5: cell activity of recombinant Serratia marcescens/pET-28a-xrpA under acid stress condition
The method comprises the following specific steps:
the recombinant Serratia marcescens Serratia marcescens/pET-28a-Vector (comparison) only containing the blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA obtained in the example 1 are respectively streaked on an LB solid culture medium and cultured for 16h at the temperature of 30 ℃ to obtain a single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 12h under the conditions of 30 ℃ and 180rpm to obtain seed liquid; inoculating the seed solution into LB liquid culture medium with pH of 4, and controlling initial OD600Culturing at 30 ℃ and 180rpm (0.1), taking 200 μ L of LB solid medium coated with 50 μ g/mL kanamycin every two hours, controlling the number of colonies on each LB solid medium to be 30-300, culturing at 30 ℃ for 12-24 hours, counting the formed colonies and calculating the cell activity (see Table 3 for the cell activity);
cellular activity＝(M/M0)×100％；
In the formula, M0Is the number of viable colonies on LB solid medium containing 50. mu.g/mL kanamycin after 0h of culture in LB liquid medium at pH 4.0; m is the number of viable colonies grown on LB solid medium containing 50. mu.g/mL kanamycin after various times of culture in LB liquid medium at pH 4.0.
As can be seen from Table 3, after the recombinant Serratia marcescens Serratiamarcescens/pET-28a-xrpA is cultured for different times under the acid stress condition, the cell activity of the recombinant Serratia marcescens is better than that of a control group, which indicates that the acid stress causes the reduction of the cell activity of the Serratia marcescens, and the DNA binding protein XrpA can improve the cell activity of the Serratia marcescens under the acid stress.
TABLE 3 cellular activities of different recombinant Serratia marcescens after stress for different periods of time at pH 4
Example 6: cell membrane integrity of recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA under acid stress condition
The method comprises the following specific steps:
the recombinant Serratia marcescens Serratia marcescens/pET-28a-Vector (comparison) only containing the blank plasmid pET-28a (+) and the recombinant Serratia marcescens Serratia marcescens/pET-28a-xrpA obtained in the example 1 are respectively streaked on an LB solid culture medium and cultured for 16h at the temperature of 30 ℃ to obtain a single colony; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, and culturing for 12h under the conditions of 30 ℃ and 180rpm to obtain seed liquid; respectively inoculating the seed liquid into LB liquid culture medium, culturing at 30 deg.C and 180rpm to OD600Obtaining a culture solution as 1; the culture medium was centrifuged to collect cells, and PBS buffer (NaCl 8.0g/L, KH) was used2PO40.2g/L、Na2HPO4·H2O2.9 g/L, KCl 0.2.2 g/L) for 2 times, and respectively suspending into an LB liquid culture medium with the same volume or an LB liquid culture medium with the pH value of 4.0 for treatment for 4 hours to obtain a bacterial liquid to be detected; OD of bacterial liquid to be measured600Is adjusted to 0.8, sucking 500 mu L of the suspension, washing the suspension for 2 times by using PBS buffer solution, collecting thalli, and then re-suspending the thalli by using 500 mu LPBS buffer solution to obtain a re-suspension A; adding 5 mu LPI dye solution into the heavy suspension A, immediately reacting for 5min in a dark place, centrifugally collecting thalli, washing for 2 times by using PBS buffer solution, collecting thalli, and then re-suspending by using 500 mu LPBS buffer solution to obtain a heavy suspension B; heavy suspension B was detected by flow cytometry, 10000 cells in the sample were analyzed at low flow rate, and data was analyzed for cell membrane integrity using FlowJo software.
The analysis finds that:
(1) in LB liquid culture medium, the cell membrane integrity of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA are 93.6% and 96.1% respectively, which shows that the cell integrity of the two strains is not damaged under normal conditions;
(2) in LB liquid medium with pH 4.0, the cell membrane integrity of recombinant Serratia marcescens/pET-28a-Vector (control) and recombinant Serratia marcescens/pET-28a-xrpA are 81.6% and 87.9% respectively, which shows that acid stress causes the reduction of the cell membrane integrity of Serratia marcescens, and DNA binding protein XrpA can improve the cell membrane integrity under the acid stress.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> method for improving acid stress resistance of Serratia marcescens
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acgcttatcc tgcggcagcc gcaactgggt attctcgcgc tgcgcctcgc ccaactcatt 360
gatcagcacc gccagcaaat cgcgcgcgtg gggctgtgcc aacgcatagt ccagcggcca 420
gtgcgcgatg cgctcgacga tcgccgccaa cagcgggttt accgccagca ccgttgcctg 480
tggcggcatg gcgggcgtca gcgccggatc gaaatacagc gagcgatacg cgacgcggcc 540
gcgcagctgc acccgatgcg caatgccgcc cggtatccag acgctgcgcg tcggcggcag 600
aatcagccac tgctgcgcca acgtgaccgt catgcacccc tgcggcgcat acagcagctg 660
cgaacgctga tgccaatgca gcccggagtc gtgatccgcc agtgccgccg cgatgccgag 720
agccggtgcc ggcagccgat ccgggtcgaa cgggtgatgc tgttgaatca gcgccat 777
Claims (10)
1. A method for improving the acid stress resistance of Serratia marcescens is characterized in that the method comprises the steps of over-expressing DNA binding protein XrpA in the Serratia marcescens; the DNA binding protein XrpA is a protein on the cell membrane of microorganisms for recognizing and binding DNA.
2. The method for improving acid stress resistance of Serratia marcescens according to claim 1, wherein the DNA binding protein XrpA has the amino acid sequence shown in SEQ ID No. 1.
3. The method for improving the acid stress resistance of Serratia marcescens according to claim 1 or 2, wherein the nucleotide sequence of the gene encoding the DNA binding protein XrpA is shown in SEQ ID No. 2.
4. The method for improving acid stress resistance of Serratia marcescens according to any one of claims 1-3, wherein the overexpression is performed by ligating the gene encoding the DNA binding protein XrpA with an expression vector to construct a recombinant plasmid containing the gene encoding the DNA binding protein XrpA, and introducing the recombinant plasmid into Serratia marcescens.
5. The method for improving acid stress resistance of Serratia marcescens according to claim 4, wherein the expression vector is pET-28a (+) plasmid.
6. The method for improving acid stress resistance of Serratia marcescens according to claim 5, wherein the overexpression comprises performing double digestion on the gene coding for the DNA binding protein XrpA and the plasmid pET-28a (+) by Ecor I and BamH I to obtain a digestion product, then connecting the digestion product to construct a recombinant plasmid containing the gene coding for the DNA binding protein XrpA, and finally introducing the recombinant plasmid into the Serratia marcescens.
7. A recombinant Serratia marcescens prepared by the method of any one of claims 1-6 for improving acid stress resistance of Serratia marcescens.
8. A method for producing prodigiosin, which comprises the steps of inoculating the recombinant Serratia marcescens disclosed in claim 7 into a fermentation medium for culture to obtain a fermentation broth, and extracting prodigiosin from the fermentation broth.
9. A method for producing 2, 3-butanediol, characterized in that the method comprises inoculating the recombinant Serratia marcescens of claim 7 into a fermentation medium, culturing to obtain a fermentation broth, and extracting 2, 3-butanediol from the fermentation broth.
10. Use of a method of increasing the acid stress resistance of serratia marcescens according to any one of claims 1 to 6 or a recombinant serratia marcescens according to claim 7 or a prodigiosin-producing method according to claim 8 or a 2, 3-butanediol-producing method according to claim 9 for the preparation of food, pharmaceutical and household chemical products.
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| CN113549643B (en) * | 2021-07-15 | 2023-08-08 | 江南大学 | Method for improving synthesis of prodigiosin by Serratia marcescens through overexpression of gene psrB |
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