CN116286929A - Method for improving survival rate of bacterial strain - Google Patents
Method for improving survival rate of bacterial strain Download PDFInfo
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- CN116286929A CN116286929A CN202211450956.2A CN202211450956A CN116286929A CN 116286929 A CN116286929 A CN 116286929A CN 202211450956 A CN202211450956 A CN 202211450956A CN 116286929 A CN116286929 A CN 116286929A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
The invention relates to a method for improving the survival rate of a strain, which is characterized in that the ldh gene in lactobacillus plantarum is knocked out, and then the srlD gene is introduced into the lactobacillus plantarum, so that the sorbitol yield in the lactobacillus plantarum is increased, and the freeze-drying survival rate of the lactobacillus plantarum is improved; the srlD genes comprise srlD1 genes and srlD2 genes, the nucleotide sequence of the srlD1 genes is shown as SEQ ID NO.12, and the nucleotide sequence of the srlD2 genes is shown as SEQ ID NO. 13. Compared with the prior art, the method can effectively improve the survival rate of the strain after freeze drying, can preliminarily judge the site of the sorbitol which has the protective effect on the strain, and has better reliability.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for improving the survival rate of bacterial strains.
Background
Lactobacillus plantarum is a highly versatile strain, widely existing in various niches, and is a normal member of the human intestinal microbiota. Lactobacillus plantarum is usually cultivated in a liquid culture medium, but the liquid culture has the defects of huge volume, inconvenient transportation and the like, and the activity of the strain is rapidly reduced during the storage process [1] . Freeze-drying technology has been widely used for the preservation of lactic acid bacteria, and has become one of the most effective methods for preserving microorganisms. Sorbitol has an osmotic protection effect on strains, and after electroporation treatment, some strains are restored in a recovery culture medium containing sorbitol; sorbitol is also used as a lyoprotectant before the lyophilization process in order to provide strain protection throughout the treatment; the protective effect of 10% sorbitol on Lactobacillus plantarum JH287 was at the same level as the protective effect of sucrose, but among the other 12 Lactobacillus plantarum, the freeze-drying survival rate was only increased to 15% at maximum even with sorbitol addition [2][3][4] . How to allow sorbitol to exert the maximum protective effect in the strain is thus a problem that has not been solved so far.
[1]Hammes W P,Bantleon A,Min S.Lactic acid bacteria in meat fermentation[J].FEMS Microbiology Letters,1990,87(1):165-73.
[2]Carvalho A S,Silva J,Ho P,et al.Survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in the presence of protectants[J].Biotechnology Letters,2002.
[3]Lee S B,Kim D H,Park H D.Effects of protectant and rehydration conditions on the survival rate and malolactic fermentation efficiency of freeze-dried Lactobacillus plantarum JH287[J].Applied Microbiology and Biotechnology,2016,100(18):7853-63.
[4] In the research of physiological damage and protection strategy of lactobacillus plantarum in the freeze drying process [ D ]. Shanghai; university of Shanghai, 2019.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for improving the survival rate of the strain, and the method is characterized in that a sorbitol generation path is constructed in lactobacillus plantarum cells, so that the intracellular sorbitol can improve the freeze-drying survival rate of the strain, the maximum protection effect of the sorbitol is realized, and the strain difference is avoided.
The aim of the invention can be achieved by the following technical scheme:
a method for improving the survival rate of the strain comprises the steps of knocking out the ldh gene in lactobacillus plantarum, and then introducing the srlD gene into the lactobacillus plantarum to realize the increase of the sorbitol yield in the lactobacillus plantarum, thereby improving the freeze-drying survival rate of the lactobacillus plantarum;
the srlD genes comprise srlD1 genes and srlD2 genes,
the nucleotide sequence of the srld1 gene is shown as SEQ ID NO.12,
the nucleotide sequence of the srld2 gene is shown as SEQ ID NO. 13.
Further, the ldh gene comprises a l-ldh gene and a d-ldh gene, and the nucleotide sequence of the l-ldh gene is shown in SEQ ID NO. 11; the nucleotide sequence of the d-ldh gene is shown in SEQ ID NO. 10.
Further, the srlD gene is a sorbitol-6-phosphate dehydrogenase encoding gene in a sorbitol operon.
Further, the srlD gene is derived from WCFS1 strain.
Further, the lactobacillus plantarum is lactobacillus plantarum AR113.
Further, the sorbitol is produced by the following way: the fructose-6P is catalyzed and reduced by sorbitol-6P dehydrogenase to generate sorbitol-6P, and the sorbitol-6P is oxidized to generate sorbitol.
The above further, the sorbitol production relies on NADH as a cofactor.
The method further comprises the steps of knocking out the ldh gene in lactobacillus plantarum, introducing the srlD gene into lactobacillus plantarum, and over-expressing the srlD gene on the basis of knocking out the ldh strain to generate excessive NADH auxiliary factors, reversing the metabolic reaction of sorbitol and promoting sorbitol-6P dehydrogenase to go towards the direction of sorbitol production.
Further, lactobacillus plantarum of the oversurface srld gene grows normally.
The method further comprises the steps of knocking out the ldh gene in the lactobacillus plantarum to obtain a lactobacillus plantarum mutant, generating excessive NADH for glycolysis conversion of an available carbon source by the lactobacillus plantarum mutant, activating a metabolic pathway of oxidized NADH, reversing a catabolic pathway of sorbitol utilization, introducing the srlD gene into the lactobacillus plantarum, enabling overproduction of sorbitol-6P dehydrogenase (Stl 6 PDH), enabling glycolysis flux to deviate from fructose-6P to a final product sorbitol, realizing sorbitol generation in the lactobacillus plantarum, and improving the freeze-drying survival rate of the lactobacillus plantarum.
The principle of the invention is as follows:
the industrial application of probiotics as starter cultures depends on their technical properties, concentration and freshness technology, and it is essential to maintain high viability, acidification activity and enzymatic activity during long-term storage. At present, a freeze drying method is selected to treat strains, and how to keep the high activity of the strains after the whole treatment is finished is a problem which is concerned at present.
Sorbitol is generally not produced at detectable levels in lactic acid bacteria, but wherein the strain has the ability to use sorbitol as a carbon source via the sorbitol operon. The operator possibly involved in sorbitol catabolism is identified in the genome of lactobacillus plantarum, and the srlD gene codes for sorbitol-6P-dehydrogenase (Stl 6 PDH), and the aim of increasing sorbitol yield in lactobacillus plantarum is achieved by expressing the sorbitol-6-phosphate dehydrogenase coding gene (srlD) in the operator to reverse the catabolic pathway of sorbitol utilization. The freeze-dried viability of the strain is provided by a method of intracellular sorbitol production.
Exogenous addition of sorbitol is closely related to the stress resistance of lactobacillus plantarum against freeze-drying reverse environment, but the protective effect of exogenous addition of sorbitol on strains varies greatly among strains, wherein the freeze-drying survival rate of lactobacillus plantarum can only be improved to 15% at most. There is therefore a need for a method that increases the protective effect of sorbitol on strains and reduces the variability between strains.
At present, a plurality of physical methods can effectively transfer small molecule protective agents into cells, and common transfer methods include heat shock, permeation, electric shock, liquid endocytosis, phospholipid phase change and the like; the loading time required for liquid phase endocytosis is longer and less efficient, and the conditions and time required for permeation are longer. The electroporation method used for loading small molecules into cells has great application potential. The PEF electric field has proven to be a useful method for loading protectants into bacterial strains, and in addition studies have been made to exploit the visual characteristics of PI dyes to investigate whether the cell membrane is reversible or irreversible after PEF treatment, whereas PI species have a molecular weight of 668Da, greater than that of sorbitol. Loading of the small molecule protectant sorbitol into the cells by PEF has therefore been shown to provide better intracellular protection, improving the freeze-drying survival of the strain.
Compared with the prior art, the invention has the following advantages:
1. the invention can effectively improve the survival rate of the strain after freeze drying, can preliminarily judge the site of sorbitol which has a protective effect on the strain, and has better reliability;
2. the invention provides a protective agent in cells, which has better protective effect on bacterial strains in the freeze drying process;
3. the invention carries out more intensive research from the internal aspect of the strain, analyzes the corresponding biosynthesis characteristics of the protective agent and the differences of related applications, and achieves the aim of reducing the damage of the strain in the freeze-drying process.
Drawings
FIG. 1 is a flow chart of over-expression plasmid construction;
FIG. 2 is a PEF process flow diagram;
FIG. 3 shows the relevant metabolic engineering system of intracellular sorbitol, wherein [1] is sorbitol Phosphate Transport System (PTS), [2] is lactate dehydrogenase, [3] is Stl6P dehydrogenase, and [4] is Mtl P dehydrogenase;
FIG. 4 is an electrophoretogram of the construction of an overexpressing strain;
FIG. 5 is the expression of a protein of interest for recombinant strains;
FIG. 6 shows the survival rate of recombinant strains after freeze-drying treatment;
FIG. 7 shows the freeze-dried viability of different Lactobacillus plantarum PEF treatments, wherein a is PBS medium PEF treatment (control: no sorbitol in the cell) and b is sorbitol medium PEF treatment (sorbitol in the cell);
FIG. 8 shows the freeze-dried viability of different probiotics after PEF treatment, wherein a is PBS medium PEF treatment (control: no sorbitol in the cell) and b is sorbitol medium PEF treatment (sorbitol in the cell).
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The present embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.
In the following examples, the materials required are as follows:
MRS Medium (1L): 10.0g of peptone, 5.0g of yeast powder, 10.0g of beef extract powder, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween-80, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and pH of 6.2-6.6, and is used for activating, culturing and counting lactobacillus and sterilizing at 115 ℃ for 20min;
LB medium (1L): 10.0g of tryptone, and sterilizing yeast extract powder for 15min at 5.0g,NaCl 10.0g,121 ℃;
reagent: trizol, his-tag antibody, seamless cloning enzyme, PBS buffer, sorbitol protectant.
The strains and plasmid sources are shown in table 1:
TABLE 1 strains and their characteristics
The information stored in lactobacillus plantarum AR113 is as follows: lactobacillus plantarum (Lactobacillus plantarum) AR113 has a preservation number of CGMCC No.13909, a preservation unit of China center for general microbiological culture Collection, a preservation address of North Xili No.1, 3 of the Korean area of Beijing city, and a preservation date of 2017, 03 and 22 days, and is disclosed in a CN111304134A patent.
The S-3 preservation information of Streptococcus thermophilus is as follows: streptococcus thermophilus (Streptococcus thermophilus) has a preservation number of CGMCC No.12098, a preservation unit of China general microbiological culture Collection center, a preservation address of North Xili No.1, 3 of the Korean area of Beijing city, and a preservation date of 2016 of 1 month and 22 days, which is disclosed in a CN108220201A patent.
The information on the deposit of lactobacillus plantarum AR307 is as follows: lactobacillus plantarum (Lactobacillus plantarum) has a preservation number of CGMCC No.10773, a preservation unit of China center for common microorganism strain preservation and management, a preservation address of North Chen Xili No.1 and 3 in the Korean area of Beijing city, and a preservation date of 29 days of 4 months in 2015, which is disclosed in the CN105400725A patent.
The information on the deposit of lactobacillus plantarum AR495 is as follows: lactobacillus plantarum (Lactobacillus plantarum) has a preservation number of CGMCC No.14004, a preservation unit of China general microbiological culture Collection center, a preservation address of North Chen West Lu No.1, 3 of the Korean area of Beijing city, and a preservation date of 2017, 04 month 07, and is disclosed in CN 114196600A.
Lactobacillus plantarum WCFS1 and lactococcus lactis NZ9000 are commercially available.
Lactobacillus plantarum AR514 is obtained by the inventor through self-screening in a laboratory.
In the examples below, the desired plasmids are shown below:
pIB184 plasmid was derived from the article
Shuttle expression plasmids for genetic studies in Streptococcus mutans.
Biswas I,Jha JK,Fromm N.Microbiology.2008Aug；154(Pt 8):2275-82.doi:10.1099/mic.0.2008/019265-0.
In the following examples, the required instrumentation is as follows:
PCR instrument, chemiDoc xrs+ gel imager, anaerobic incubator, vacuum freeze dryer, microPulse electrotransformation instrument.
In the following examples, the nucleotide sequences are shown below:
the nucleotide sequence of the primer srld1-F is shown as SEQ ID NO. 1:
5’-TGGATCCCCGCGGTACCCGGGAATTCTTATCCTCGCGATTTAC-3’；
the nucleotide sequence of the primer srld1-R is shown as SEQ ID NO. 2:
5’-AATCACTTAAGCTTATCGATAGATCTATGACGACAGATTGGTT-3’；
the nucleotide sequence of the primer srld2-F is shown as SEQ ID NO. 3:
5’-TGGATCCCCGCGGTACCCGGGAATTCTTAACCACGACTCTTGC-3’；
the nucleotide sequence of the primer srld2-R is shown as SEQ ID NO. 4:
5’-AATCACTTAAGCTTATCGATAGATCTATGAATAATTCATGGAT-3’；
the nucleotide sequence of the pIB184 plasmid is shown in SEQ ID NO. 5:
aactttgcaatttgttctagtgtttttatggttggatctgattttcctgattctattcgtgaataatttgatctactcatttctaattcttggggtaccgccagcatttcggaaaaaaaccacgctaaggattttttctataaaaagagccgttatattaagaataaaacggctcttttatacgtaaaggacgtaaattcatttgcccagtgtcatgtaatccttcaaatttgtattctccaagaaaattgatatgttcccatcctaacggccacgcatatggcattaaatcttctctaaattctcctcttgcttttaattcttctacggctttttccatatatacagtgttccacacacttatagcgttaataattatgtttagtgcactagctctttgtaactggtcttggagagcacgttctctaaattctccacgttgtccaaaaaatatagttctagctaatgcattgattgcttctcctttatttaaacctttttgaacccgtctccttacggctttattagatatgtaatccagcgtaaagagggttttctcgattcgtcccatttctccaagtgctgttgcgagtttattttgtcttgcatatgatccgagcttccccatgataagagcgctagggacctctttagctccttggaagctgtcagtagtatacctaataatttatctacattccctttagtaacgtgtaactttccaaatttacaaaagcgactcatagaattatttcctcccgttaaataatagataactattaaaaatagacaatacttgctcataagtaacggtacttaaattgtttactttggcgtgtttcattgcttgatgaaactgatttttagtaaacagttgacgatattctcgattgacccattttgaaacaaagtacgtatatagcttccaatatttatctggaacatctgtggtatggcgggtaagttttattaagacactgtttacttttggtttaggatgaaagcattccgctggcagcttaagcaattgctgaatcgagacttgagtgtgcaagagcaaccctagtgttcggtgaatatccaaggtacgcttgtagaatccttcttcaacaatcagatagatgtcagacgcatggctttcaaaaaccacttttttaataatttgtgtgcttaaatggtaaggaatactcccaacaattttatacctctgtttgttagggaattgaaactgtagaatatcttggtgaattaaagtgacacgagtattcagttttaatttttctgacgataagttgaatagatgactgtctaattcaatagacgttacctgtttacttattttagccagtttcgtcgttaaatgccctttacctgttccaatttcgtaaacggtatcggtttcttttaaattcaattgttttattatttggttgagtactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatcactccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaattttaagatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttcttttacgtttccgccattctttgctgtttcgatttttatgatatggtgcaagtcagcacgaacacgaaccgtcttatctcccattatatctttttttgcactgattggtgtatcatttcgtttttcttttgcgcttcttgataaaagggatagtaattcattcctggttgcaaattttgaaaaccgctacggatcacatctttttctaaactattgatccatagtcttttatacgttttatctttagaaaaggcatttgctttatgaatgatcgaccaggcaatgttttcgccttctctgtcgctatctgttgcgacaatgattgtatttgcttgttttaaaagttcagcaacaattttaaactgcttttttttatctgttgccacttcaaaatcgtatcgattctagatttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgtctagaatcgataagcttggctgcaggtcgataaggctattggtgtttatggctctcttggtcgtcagactgatgggccccgaaaagccctgacaacccttgttcctaaaaaggaataagcgttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtcttagaattaaattaaaaagggaggccaaatataatgaaaaatatgaatgacaatgatgttggatccccgcggtacccgggaattctagagctcgagatctatcgataagcttaagtgattagtcaaagaatggtgatgacaattgtaaattctatttaatcactttgactagcaaatactaacaacaagacacacacaccaaaaatcaaaaattcactacttttagttaaaaaccacgtaaccacaagaactaatccaatccatgtaatcgggttcttcaaatatttctccaagattttcctcctctaatatgctcaacttaaatgacctattcaataaatctattatgctgctaaatagtttataggacaaataagtatactctaatgacctataaaagatagaaaattaaaaaatcaagtgttcgcttcgctctcactgcccctcgacgttttagtagcctttccctcacttcgttcagtccaagccaactaaaagttttcgggctactctctccttctccccctaataattaattaaaatcttactctgtatatttctgctaatcattcgctaaacagcaaagaaaaaacaaacacgtatcatagatataaatgtaatggcatagtgcgggttttattttcagcctgtatcatagctaaacaaatcgagttgtgtgtccgttttagggcgttctgctagcttgtttaaagtctcttgaatgaatgtatgctctaagtcaaaagaatttgtcagcgcctttatatagctttctttttcttctttttttactttaatgatcgatagcaacaatgatttaacactagcaagttgaatgccaccatttcttcctggtttaatcttaaagaaaatttcctgattcgccttcagtaccttcagcaatttatctaatgtccgttcaggaatgcctagcacttctctaatctcttttttggtcgtcactaaataaggcttgtatacatcgcttttttcgctaatataagccattaaatcttctttccattctgacaaatgaacacgttgacgttcgcttctttttttcttgaatttaaaccacccttgacggacaaataaatctttactggttaaatcacttgatacccaagctttgcaaagaatggtaatgtattccctattagccccttgatagttttctgaataggcacttctaacaattttgattacttctttttcttctaagggttgatctaatcgattattaaactcaaacatattatattcgcacgtttcgattgaatagcctgaactaaagtaggctaaagagagggtaaacatgacgttattacgccctattaaacccttttctcctgaaaatttcgtttcgtgcaataagagattaaaccagggttcatctacttgttttttgccttctgtaccgcttaaaaccgttagacttgaacgagtaaagcccttattatctgtttgtttgaaagaccaatcttgccattctttgaaagaataacggtaattaggatcaaaaaattctacattgtccgttcttggtatgcgagcaataccaaaatgattacacgttagatcaactggcaaagactttccaaaatattctcggatattttgcgaaattattttggctgctttgacagatttaaattctgattttgaagtcacatagactggcgtttctaaaacaaaatatgcttgataacctttatcagatttgataatcatagtaggcataaaacctaaatcaatagcggttgttaaaatatcgcttgctgaaatagtttcttttgccgtgtgaatatcaaaatcaataaagaaggtattgatttgtcttaaattgttttcagaatgtcctttcgtgtatgaacggttttcgtctgcatacgttccataacgataaacgttgggtgtccaatgtgtaaatgtatcttgattttcttgaatcgcttcctcggaagtcagaacaacaccacgaccgccaatcatgcttgattttgagcgatacgcaaaaatagcccctttgcttttacctggcttggtagtgattgagcgaattttactatttttaaatttgtactttaacaagccgtcatgaagcacagtttctacaacaaaagggatattcattcagctgttctcctttcctataaatcctataaaataggttgtttaattaacttggtttgctttttcattcaactgtttcaatattgcatgttttgaaaaagatttttttcctttataagtcaatttttttccactaatcgaataaattattttgttattttctattaacttatatatataatcttccccctccgaagaaaaatacttatctgattttgtttctaagtagatatttctcttttctaactctttcttaaacgtttctagtgtatagatatttgctaattttcttatctccaataaactattttttatataagttttacattcatcatgattcatacaaactccaccttctataaatgaatacaaaaaaagcaatcaaacgatttccgattgattgcttaacaattcttaaattcagtagcttagatacttgaaaactctctgatttccctatataatgatagtacggttatataccgtcttcaaacaaagttaattaaataacttcttacgagggaagagttcatctgactaactgataagcgttggtttggcaatcttatcgggctatgcatttataaaatgtcgtcaaacattttataaatgtgtcatggctcttttttcgtttctattcagttcgttgtttcgttatatctagtataccgcttttaaaaaaaaataagcaacgatttcgtgcattattcacacgaagtcattgcttttttcttcttccatttctaaatccaatgttacttgttctgattctgtttctggctctggttctgttggctcatttgggattaaatccactactagcgttgagttagtt；
the nucleotide sequence of the primer pIB184-yz-F is shown in SEQ ID NO. 6:
5’-GATGGGCCCCGAAAAGC-3’；
the nucleotide sequence of the primer pIB184-yz-R is shown in SEQ ID NO. 7:
5’-GAGAAATATTTGAAGAACCCGATTACATGG-3’；
the nucleotide sequence of the plasmid pIB184-srld1 is shown in SEQ ID NO. 8:
aactttgcaatttgttctagtgtttttatggttggatctgattttcctgattctattcgtgaataatttgatctactcatttctaattcttggggtaccgccagcatttcggaaaaaaaccacgctaaggattttttctataaaaagagccgttatattaagaataaaacggctcttttatacgtaaaggacgtaaattcatttgcccagtgtcatgtaatccttcaaatttgtattctccaagaaaattgatatgttcccatcctaacggccacgcatatggcattaaatcttctctaaattctcctcttgcttttaattcttctacggctttttccatatatacagtgttccacacacttatagcgttaataattatgtttagtgcactagctctttgtaactggtcttggagagcacgttctctaaattctccacgttgtccaaaaaatatagttctagctaatgcattgattgcttctcctttatttaaacctttttgaacccgtctccttacggctttattagatatgtaatccagcgtaaagagggttttctcgattcgtcccatttctccaagtgctgttgcgagtttattttgtcttgcatatgatccgagcttccccatgataagagcgctagggacctctttagctccttggaagctgtcagtagtatacctaataatttatctacattccctttagtaacgtgtaactttccaaatttacaaaagcgactcatagaattatttcctcccgttaaataatagataactattaaaaatagacaatacttgctcataagtaacggtacttaaattgtttactttggcgtgtttcattgcttgatgaaactgatttttagtaaacagttgacgatattctcgattgacccattttgaaacaaagtacgtatatagcttccaatatttatctggaacatctgtggtatggcgggtaagttttattaagacactgtttacttttggtttaggatgaaagcattccgctggcagcttaagcaattgctgaatcgagacttgagtgtgcaagagcaaccctagtgttcggtgaatatccaaggtacgcttgtagaatccttcttcaacaatcagatagatgtcagacgcatggctttcaaaaaccacttttttaataatttgtgtgcttaaatggtaaggaatactcccaacaattttatacctctgtttgttagggaattgaaactgtagaatatcttggtgaattaaagtgacacgagtattcagttttaatttttctgacgataagttgaatagatgactgtctaattcaatagacgttacctgtttacttattttagccagtttcgtcgttaaatgccctttacctgttccaatttcgtaaacggtatcggtttcttttaaattcaattgttttattatttggttgagtactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatcactccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaattttaagatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttcttttacgtttccgccattctttgctgtttcgatttttatgatatggtgcaagtcagcacgaacacgaaccgtcttatctcccattatatctttttttgcactgattggtgtatcatttcgtttttcttttgcgcttcttgataaaagggatagtaattcattcctggttgcaaattttgaaaaccgctacggatcacatctttttctaaactattgatccatagtcttttatacgttttatctttagaaaaggcatttgctttatgaatgatcgaccaggcaatgttttcgccttctctgtcgctatctgttgcgacaatgattgtatttgcttgttttaaaagttcagcaacaattttaaactgcttttttttatctgttgccacttcaaaatcgtatcgattctagatttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgtctagaatcgataagcttggctgcaggtcgataaggctattggtgtttatggctctcttggtcgtcagactgatgggccccgaaaagccctgacaacccttgttcctaaaaaggaataagcgttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtcttagaattaaattaaaaagggaggccaaatataatgaaaaatatgaatgacaatgatgttggatccccgcggtacccgggaattcatgacgacagattggttgggcttaaaagacaaaatcgttcttgttacgggtggctcgtctggaattggtgatgcgattgtcgaatcattattgaataacggtgcaaaggttgtcgatttagatttacattcgagttctaggaagaaccaacactttgattttgttgagtgtgatgtatcgtctgaagaggcagtgacggaggcaatagaccagattttagataagtttggccggattgatgggctggttaataatgctggcataaacttaccaaggttattggttgatgagcagatgccacgtggacaatatgagttatgtacggaagtctttaataagatgtttgctgtaaatgttagaagtactttcttaatgtcacaaacggtaggtcgacagatggtgaagcaacggtcgggaactattgtaaacatgtcatcagaagcaggactagaagggtcccaggggcagagtgtctatgccgcaaccaaaggggcgattaatggcctgactcgttcttggtcgaaagaactaggggtattcaatattagagtggttggggtcgcacctggcattatggatgcaactggattgcggacgccatcctatgaagaggcgttagcgtattcacggcatcaaacagttcaacaattacgatcaggttacacaagcaaaagtactacacctttagggcgaagcggacatttatatgaagttgcagatttggttagctatttactgtctgaacgggcaagttatattactggtatcactgttaatgtggcaggcggtaaatcgcgaggataatagagctcgagatctatcgataagcttaagtgattagtcaaagaatggtgatgacaattgtaaattctatttaatcactttgactagcaaatactaacaacaagacacacacaccaaaaatcaaaaattcactacttttagttaaaaaccacgtaaccacaagaactaatccaatccatgtaatcgggttcttcaaatatttctccaagattttcctcctctaatatgctcaacttaaatgacctattcaataaatctattatgctgctaaatagtttataggacaaataagtatactctaatgacctataaaagatagaaaattaaaaaatcaagtgttcgcttcgctctcactgcccctcgacgttttagtagcctttccctcacttcgttcagtccaagccaactaaaagttttcgggctactctctccttctccccctaataattaattaaaatcttactctgtatatttctgctaatcattcgctaaacagcaaagaaaaaacaaacacgtatcatagatataaatgtaatggcatagtgcgggttttattttcagcctgtatcatagctaaacaaatcgagttgtgtgtccgttttagggcgttctgctagcttgtttaaagtctcttgaatgaatgtatgctctaagtcaaaagaatttgtcagcgcctttatatagctttctttttcttctttttttactttaatgatcgatagcaacaatgatttaacactagcaagttgaatgccaccatttcttcctggtttaatcttaaagaaaatttcctgattcgccttcagtaccttcagcaatttatctaatgtccgttcaggaatgcctagcacttctctaatctcttttttggtcgtcactaaataaggcttgtatacatcgcttttttcgctaatataagccattaaatcttctttccattctgacaaatgaacacgttgacgttcgcttctttttttcttgaatttaaaccacccttgacggacaaataaatctttactggttaaatcacttgatacccaagctttgcaaagaatggtaatgtattccctattagccccttgatagttttctgaataggcacttctaacaattttgattacttctttttcttctaagggttgatctaatcgattattaaactcaaacatattatattcgcacgtttcgattgaatagcctgaactaaagtaggctaaagagagggtaaacatgacgttattacgccctattaaacccttttctcctgaaaatttcgtttcgtgcaataagagattaaaccagggttcatctacttgttttttgccttctgtaccgcttaaaaccgttagacttgaacgagtaaagcccttattatctgtttgtttgaaagaccaatcttgccattctttgaaagaataacggtaattaggatcaaaaaattctacattgtccgttcttggtatgcgagcaataccaaaatgattacacgttagatcaactggcaaagactttccaaaatattctcggatattttgcgaaattattttggctgctttgacagatttaaattctgattttgaagtcacatagactggcgtttctaaaacaaaatatgcttgataacctttatcagatttgataatcatagtaggcataaaacctaaatcaatagcggttgttaaaatatcgcttgctgaaatagtttcttttgccgtgtgaatatcaaaatcaataaagaaggtattgatttgtcttaaattgttttcagaatgtcctttcgtgtatgaacggttttcgtctgcatacgttccataacgataaacgttgggtgtccaatgtgtaaatgtatcttgattttcttgaatcgcttcctcggaagtcagaacaacaccacgaccgccaatcatgcttgattttgagcgatacgcaaaaatagcccctttgcttttacctggcttggtagtgattgagcgaattttactatttttaaatttgtactttaacaagccgtcatgaagcacagtttctacaacaaaagggatattcattcagctgttctcctttcctataaatcctataaaataggttgtttaattaacttggtttgctttttcattcaactgtttcaatattgcatgttttgaaaaagatttttttcctttataagtcaatttttttccactaatcgaataaattattttgttattttctattaacttatatatataatcttccccctccgaagaaaaatacttatctgattttgtttctaagtagatatttctcttttctaactctttcttaaacgtttctagtgtatagatatttgctaattttcttatctccaataaactattttttatataagttttacattcatcatgattcatacaaactccaccttctataaatgaatacaaaaaaagcaatcaaacgatttccgattgattgcttaacaattcttaaattcagtagcttagatacttgaaaactctctgatttccctatataatgatagtacggttatataccgtcttcaaacaaagttaattaaataacttcttacgagggaagagttcatctgactaactgataagcgttggtttggcaatcttatcgggctatgcatttataaaatgtcgtcaaacattttataaatgtgtcatggctcttttttcgtttctattcagttcgttgtttcgttatatctagtataccgcttttaaaaaaaaataagcaacgatttcgtgcattattcacacgaagtcattgcttttttcttcttccatttctaaatccaatgttacttgttctgattctgtttctggctctggttctgttggctcatttgggattaaatccactactagcgttgagttagtt；
the nucleotide sequence of the plasmid pIB184-srld2 is shown in SEQ ID NO. 9:
aactttgcaatttgttctagtgtttttatggttggatctgattttcctgattctattcgtgaataatttgatctactcatttctaattcttggggtaccgccagcatttcggaaaaaaaccacgctaaggattttttctataaaaagagccgttatattaagaataaaacggctcttttatacgtaaaggacgtaaattcatttgcccagtgtcatgtaatccttcaaatttgtattctccaagaaaattgatatgttcccatcctaacggccacgcatatggcattaaatcttctctaaattctcctcttgcttttaattcttctacggctttttccatatatacagtgttccacacacttatagcgttaataattatgtttagtgcactagctctttgtaactggtcttggagagcacgttctctaaattctccacgttgtccaaaaaatatagttctagctaatgcattgattgcttctcctttatttaaacctttttgaacccgtctccttacggctttattagatatgtaatccagcgtaaagagggttttctcgattcgtcccatttctccaagtgctgttgcgagtttattttgtcttgcatatgatccgagcttccccatgataagagcgctagggacctctttagctccttggaagctgtcagtagtatacctaataatttatctacattccctttagtaacgtgtaactttccaaatttacaaaagcgactcatagaattatttcctcccgttaaataatagataactattaaaaatagacaatacttgctcataagtaacggtacttaaattgtttactttggcgtgtttcattgcttgatgaaactgatttttagtaaacagttgacgatattctcgattgacccattttgaaacaaagtacgtatatagcttccaatatttatctggaacatctgtggtatggcgggtaagttttattaagacactgtttacttttggtttaggatgaaagcattccgctggcagcttaagcaattgctgaatcgagacttgagtgtgcaagagcaaccctagtgttcggtgaatatccaaggtacgcttgtagaatccttcttcaacaatcagatagatgtcagacgcatggctttcaaaaaccacttttttaataatttgtgtgcttaaatggtaaggaatactcccaacaattttatacctctgtttgttagggaattgaaactgtagaatatcttggtgaattaaagtgacacgagtattcagttttaatttttctgacgataagttgaatagatgactgtctaattcaatagacgttacctgtttacttattttagccagtttcgtcgttaaatgccctttacctgttccaatttcgtaaacggtatcggtttcttttaaattcaattgttttattatttggttgagtactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatcactccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaattttaagatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttcttttacgtttccgccattctttgctgtttcgatttttatgatatggtgcaagtcagcacgaacacgaaccgtcttatctcccattatatctttttttgcactgattggtgtatcatttcgtttttcttttgcgcttcttgataaaagggatagtaattcattcctggttgcaaattttgaaaaccgctacggatcacatctttttctaaactattgatccatagtcttttatacgttttatctttagaaaaggcatttgctttatgaatgatcgaccaggcaatgttttcgccttctctgtcgctatctgttgcgacaatgattgtatttgcttgttttaaaagttcagcaacaattttaaactgcttttttttatctgttgccacttcaaaatcgtatcgattctagatttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgtctagaatcgataagcttggctgcaggtcgataaggctattggtgtttatggctctcttggtcgtcagactgatgggccccgaaaagccctgacaacccttgttcctaaaaaggaataagcgttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtcttagaattaaattaaaaagggaggccaaatataatgaaaaatatgaatgacaatgatgttggatccccgcggtacccgggaattcatgaataattcatggattaatatttcgggtaaggtatacgttgttactgggggctcatccggtattggtaaagcaattgtaactgaattattgaataacggggcgattgtttataatgctgacctcaatcaaggtgatcagcaaaatgataatttacactttgttgaaaccaatgtcacagatgcggaagcggttaatcggctggtcttaaaggttgttgatgaacaaggaaaaattgacgggcttgtaaataatgctggtatcaatttgccacgtttattggctgacgccaaggatcctcacggtaagtatgaatttgcagaatcggattttgtgaaaatgtttgccgtgaacgtgaaaagcgtcttccttgtatcgcaggcagttacccatcaattcgagaagcaacagtacggtgttattgttaatatgtcctcagaagctggacttgaaggttcacaaggacagagtgtttattcagcaactaagggagcgattaatgggttcactcgttcgtgggctaaagaattaggtgaacataatattcgggttgttggtgtcgcgccaggtattatggaagctactgggctaagaacacgctcttatgaagaggcgttagcttatacccggcataaaactgttgatgacattcgggcaggctataagagtacgtcaaccacacctcttggtcggagtggcaaactatcagaagttgcggatgcagttaactatttactatcagaacgtgccagttacattaccggggttacgattaacgttgcgggaggcaagagtcgtggttaatagagctcgagatctatcgataagcttaagtgattagtcaaagaatggtgatgacaattgtaaattctatttaatcactttgactagcaaatactaacaacaagacacacacaccaaaaatcaaaaattcactacttttagttaaaaaccacgtaaccacaagaactaatccaatccatgtaatcgggttcttcaaatatttctccaagattttcctcctctaatatgctcaacttaaatgacctattcaataaatctattatgctgctaaatagtttataggacaaataagtatactctaatgacctataaaagatagaaaattaaaaaatcaagtgttcgcttcgctctcactgcccctcgacgttttagtagcctttccctcacttcgttcagtccaagccaactaaaagttttcgggctactctctccttctccccctaataattaattaaaatcttactctgtatatttctgctaatcattcgctaaacagcaaagaaaaaacaaacacgtatcatagatataaatgtaatggcatagtgcgggttttattttcagcctgtatcatagctaaacaaatcgagttgtgtgtccgttttagggcgttctgctagcttgtttaaagtctcttgaatgaatgtatgctctaagtcaaaagaatttgtcagcgcctttatatagctttctttttcttctttttttactttaatgatcgatagcaacaatgatttaacactagcaagttgaatgccaccatttcttcctggtttaatcttaaagaaaatttcctgattcgccttcagtaccttcagcaatttatctaatgtccgttcaggaatgcctagcacttctctaatctcttttttggtcgtcactaaataaggcttgtatacatcgcttttttcgctaatataagccattaaatcttctttccattctgacaaatgaacacgttgacgttcgcttctttttttcttgaatttaaaccacccttgacggacaaataaatctttactggttaaatcacttgatacccaagctttgcaaagaatggtaatgtattccctattagccccttgatagttttctgaataggcacttctaacaattttgattacttctttttcttctaagggttgatctaatcgattattaaactcaaacatattatattcgcacgtttcgattgaatagcctgaactaaagtaggctaaagagagggtaaacatgacgttattacgccctattaaacccttttctcctgaaaatttcgtttcgtgcaataagagattaaaccagggttcatctacttgttttttgccttctgtaccgcttaaaaccgttagacttgaacgagtaaagcccttattatctgtttgtttgaaagaccaatcttgccattctttgaaagaataacggtaattaggatcaaaaaattctacattgtccgttcttggtatgcgagcaataccaaaatgattacacgttagatcaactggcaaagactttccaaaatattctcggatattttgcgaaattattttggctgctttgacagatttaaattctgattttgaagtcacatagactggcgtttctaaaacaaaatatgcttgataacctttatcagatttgataatcatagtaggcataaaacctaaatcaatagcggttgttaaaatatcgcttgctgaaatagtttcttttgccgtgtgaatatcaaaatcaataaagaaggtattgatttgtcttaaattgttttcagaatgtcctttcgtgtatgaacggttttcgtctgcatacgttccataacgataaacgttgggtgtccaatgtgtaaatgtatcttgattttcttgaatcgcttcctcggaagtcagaacaacaccacgaccgccaatcatgcttgattttgagcgatacgcaaaaatagcccctttgcttttacctggcttggtagtgattgagcgaattttactatttttaaatttgtactttaacaagccgtcatgaagcacagtttctacaacaaaagggatattcattcagctgttctcctttcctataaatcctataaaataggttgtttaattaacttggtttgctttttcattcaactgtttcaatattgcatgttttgaaaaagatttttttcctttataagtcaatttttttccactaatcgaataaattattttgttattttctattaacttatatatataatcttccccctccgaagaaaaatacttatctgattttgtttctaagtagatatttctcttttctaactctttcttaaacgtttctagtgtatagatatttgctaattttcttatctccaataaactattttttatataagttttacattcatcatgattcatacaaactccaccttctataaatgaatacaaaaaaagcaatcaaacgatttccgattgattgcttaacaattcttaaattcagtagcttagatacttgaaaactctctgatttccctatataatgatagtacggttatataccgtcttcaaacaaagttaattaaataacttcttacgagggaagagttcatctgactaactgataagcgttggtttggcaatcttatcgggctatgcatttataaaatgtcgtcaaacattttataaatgtgtcatggctcttttttcgtttctattcagttcgttgtttcgttatatctagtataccgcttttaaaaaaaaataagcaacgatttcgtgcattattcacacgaagtcattgcttttttcttcttccatttctaaatccaatgttacttgttctgattctgtttctggctctggttctgttggctcatttgggattaaatccactactagcgttgagttagtt；
the nucleotide sequence of the d-ldh gene is shown in SEQ ID NO. 10:
atgaaaattattgcatatgctgtacgtgatgacgaacgtccattcttcgatacttggatgaaagaaaacccagatgttgaagttaaattagttccagaattacttactgaagacaacgttgacttagctaaaggcttcgacggtgccgatgtataccaacaaaaggactatactgctgaagtattgaacaagttagccgacgaaggggttaagaacatctctcttcgtaacgttggtgttgataacttggacgttcctactgttaaagcacgtggcttaaacatttctaacgtacctgcatactcaccaaatgcgattgctgaattatcagtaacgcaattgatgcaattattacgtcaaaccccattgttcaacaagaagttagctaagcaagacttccgttgggcaccagatattgccaaggaattaaacaccatgactgttggtgttatcggtactggtcggattggccgtgctgccatcgatattttcaaaggcttcggcgctaaggttatcggttacgatgtttaccggaatgctgaacttgaaaaggaaggcatgtacgttgacaccttggacgaattatacgcccaagctgatgttatcacgttacacgttcctgcattgaaggataactaccacatgttgaatgcggatgccttcagcaagatgaaagatggcgcctacatcttgaactttgctcgtgggacactcatcgattcagaagacttgatcaaagccttagacagtggcaaagttgccggtgccgctcttgatacgtatgaatacgaaactaagatcttcaacaaagaccttgaaggtcaaacgattgatgacaaggtcttcatgaacttgttcaaccgcgacaatgttttgattacaccacatacggctttctacactgaaactgccgttcacaacatggtgcacgtttcaatgaacagtaacaaacaattcatcgaaactggtaaagctgatacgcaagttaagtttgactaa；
the nucleotide sequence of the l-ldh gene is shown in SEQ ID NO. 11:
ttgtcaagcatgccaaatcatcaaaaagttgtgttagtcggcgacggcgctgttggttctagttacgcttttgccatggcacaacaaggaattgctgaagaatttgtaattgtcgatgttgttaaagatcggacaaagggtgacgcccttgatcttgaagacgcccaagcattcaccgctcccaagaagatttactcaggcgaatattcagattgtaaggacgctgacttagttgttattacagccggtgcgcctcaaaagcctggtgaatcacgtttagacttagttaacaagaacttaaatatcctatcatccattgtcaaaccagttgttgactccggctttgacggcatcttcttagttgctgctaaccctgttgacatcttaacttacgctacttggaaattctcaggtttcccaaaggatcgtgtcattggttcagggacttccttagactcttcacgtttacgcgttgcgttaggcaaacaattcaatgttgatcctcgttccgttgatgcttacatcatgggtgaacacggtgattctgaatttgctgcttactcaactgcaaccatcgggacacgtccagttcgcgatgtcgctaaggaacaaggcgtttctgacgaagatttagccaagttagaagatggtgttcgtaacaaagcttacgacatcatcaacttgaagggtgccacgttctacggtatcgggactgctttaatgcggatttccaaagccattttacgtgatgaaaatgccgttttaccagtaggtgcctacatggacggccaatacggcttaaacgacatttatatcgggactccggctgtgattggtggaactggtttgaaacaaatcatcgaatcaccactttcagctgacgaactcaagaagatgcaagattccgccgcaactttgaaaaaagtgcttaacgacggtttagctgaattagaaaataaataa；
the nucleotide sequence of the srld1 gene is shown in SEQ ID NO. 12:
ttatcctcgcgatttaccgcctgccacattaacagtgataccagtaatataacttgcccgttcagacagtaaatagctaaccaaatctgcaacttcatataaatgtccgcttcgccctaaaggtgtagtacttttgcttgtgtaacctgatcgtaattgttgaactgtttgatgccgtgaatacgctaacgcctcttcataggatggcgtccgcaatccagttgcatccataatgccaggtgcgaccccaaccactctaatattgaatacccctagttctttcgaccaagaacgagtcaggccattaatcgcccctttggttgcggcatagacactctgcccctgggacccttctagtcctgcttctgatgacatgtttacaatagttcccgaccgttgcttcaccatctgtcgacctaccgtttgtgacattaagaaagtacttctaacatttacagcaaacatcttattaaagacttccgtacataactcatattgtccacgtggcatctgctcatcaaccaataaccttggtaagtttatgccagcattattaaccagcccatcaatccggccaaacttatctaaaatctggtctattgcctccgtcactgcctcttcagacgatacatcacactcaacaaaatcaaagtgttggttcttcctagaactcgaatgtaaatctaaatcgacaacctttgcaccgttattcaataatgattcgacaatcgcatcaccaattccagacgagccacccgtaacaagaacgattttgtcttttaagcccaaccaatctgtcgtcat；
the nucleotide sequence of the srld2 gene is shown as SEQ ID NO. 13:
ttaaccacgactcttgcctcccgcaacgttaatcgtaaccccggtaatgtaactggcacgttctgatagtaaatagttaactgcatccgcaacttctgatagtttgccactccgaccaagaggtgtggttgacgtactcttatagcctgcccgaatgtcatcaacagttttatgccgggtataagctaacgcctcttcataagagcgtgttcttagcccagtagcttccataatacctggcgcgacaccaacaacccgaatattatgttcacctaattctttagcccacgaacgagtgaacccattaatcgctcccttagttgctgaataaacactctgtccttgtgaaccttcaagtccagcttctgaggacatattaacaataacaccgtactgttgcttctcgaattgatgggtaactgcctgcgatacaaggaagacgcttttcacgttcacggcaaacattttcacaaaatccgattctgcaaattcatacttaccgtgaggatccttggcgtcagccaataaacgtggcaaattgataccagcattatttacaagcccgtcaatttttccttgttcatcaacaacctttaagaccagccgattaaccgcttccgcatctgtgacattggtttcaacaaagtgtaaattatcattttgctgatcaccttgattgaggtcagcattataaacaatcgccccgttattcaataattcagttacaattgctttaccaataccggatgagcccccagtaacaacgtataccttacccgaaatattaatccatgaattattcat。
the remainder, unless specifically stated, is indicative of a conventional commercially available feedstock or conventional processing technique in the art.
Example 1
1.1 construction of the overexpression plasmid
Construction of Srld overexpression plasmid: the WCFS1 strain is used as a template, corresponding fragment specific primers srld1-F, srld1-R and srld2-F, srld-R are used for PCR amplification reaction, and then the kit is used for purification and recovery, wherein the fragment sizes are respectively srld1:804bp and srld2:804bp. The pIB184 plasmid is subjected to double digestion for 3 hours at 37 ℃ by using restriction enzymes BgLII and EcoRI, the purified and recovered vector and DNA fragment are subjected to seamless cloning, a seamless cloning system is reacted for 30 minutes at 37 ℃, then the seamless cloning system is transferred into E.coil Top10 to perform competent heat shock for 90 seconds, the seamless cloning system is coated on a flat plate with erythromycin final concentration of 400ng/mL, after incubation for 24 hours, single strains are selected and verified by using primers pIB184-yz-F and pIB184-yz-R, the target bands are about 1000bp, and the plasmid electronic cloning flow is named as pIB184-srld1 and pIB184-srld2 after sequencing is successful.
1.2 construction of recombinant strains
Electrotransformation of recombinant plasmids pIB184-srld1 and pIB184-srld2 into AR113 competence, and Em picking r The positive clones screened by the resistance plate are subjected to PCR verification by using verification primers pIB184-yz-F and pIB184-yz-R, sequencing is successful, the correct over-expression strains are named as L.plantarum AR113-srld1 and L.plantarum AR113-srld2, and the specific steps of electric transformation are as follows: transferring 0.1-1 mug plasmid into a competent state, standing on ice for 10min, transferring into a 2mm electric rotating cup (Bio-rad) which is pre-cooled for 40min in advance of sterilization by using ultraviolet wind, carrying out electric rotation under the conditions of 2.5kV,400 omega and 25 mu F, immediately adding 900 mu L of resuscitation liquid, standing at 37 ℃ for resuscitation for 3h, centrifuging the strain for 3min under the condition of 4500rpm, removing 700 mu L of supernatant, coating on a corresponding resistant flat plate, and carrying out anaerobic culture at 37 ℃ for 24-48 h; the same procedure was followed to transfer recombinant plasmids pIB184-srld1 and pIB184-srld2 into AR113 Δldh competence, and the correct single-gene knockout strain was obtained and designated L.plantarum AR113 Δldh-srld1 and L.plantarum AR113 Δldh-srld2.
1.3 PEF loading sorbitol into cells
OD will be cultured overnight 600 2mL of bacterial mud is centrifuged out from bacterial liquid about 1, and the bacterial mud is washed twice by normal saline and resuspended in 10% sorbitol solution (control group is resuspended in PBS), and the whole experimental flow is shown in FIG. 2. After placing an electric rotating cup in an ultra-clean bench and blowing ultraviolet light for 30min, respectively adding 400 mu L of samples into the electric rotating cup, and performing electric shock by using Gene-Pulser Xcell equipment, wherein parameters are set to 1500V voltage, two pulse durations of 0.1ms, square wave pulses and pulse interval time: PEF treatment was performed at room temperature (25 ℃ ±1 ℃). After the treatment, the samples were placed in an incubator at 37℃for resealing for 30min. Samples before and after the treatment were sampled and counted, respectively.
1.4 determination of the Freeze-drying survival of Strain
The treated bacterial sludge was resuspended in 1mL sorbitol protectant (PBS control), then transferred to penicillin bottles and placed in a freeze dryer with the program set for freeze drying as follows: firstly, pre-freezing the sample at the temperature of minus 40 ℃ for 3 hours, then heating to minus 30 ℃ at the speed of 1 ℃/min for drying for 800 minutes, and then raising the temperature to 25 ℃ at the speed of 1 ℃/min for secondary drying for 2 hours. Wherein the cold trap temperature is about-80 ℃ and the vacuum degree is 20Pa. Counting the bacterial powder after freeze drying by using a gradient dilution counting method, sucking 10 mu L of diluent for plate counting, and placing the plate at 37 ℃ for culturing for 36-48 hours. The freeze-drying survival rate is as follows: number of viable bacteria after lyophilization/number of viable bacteria before lyophilization.
Example 2 results
2.1 construction of Lactobacillus plantarum producing sorbitol intracellular
The ldh-deficient lactobacillus plantarum mutant produces excess NADH for glycolytic conversion of available carbon sources, dissipates by activating metabolic pathways capable of oxidizing NADH to maintain balanced redox balance, and furthermore the high flux of glycolysis in the non-ldh knocked-out strain results in limited availability of the glycolytic intermediate fructose-6P, which is the substrate of the primary glycolytic reaction leading to sorbitol production. Intracellular sorbitol production is achieved by reversing the sorbitol catabolic pathway based on knockout of the l-ldh and d-ldh strains, over-expressing the srld gene such that sorbitol-6P dehydrogenase is overproduced, allowing the glycolytic flux to shift from fructose-6P to the final product sorbitol, while adding the additional NADH provided by the ldh gene knockout (see FIG. 3 for related metabolic engineering pathways).
In order to realize the efficient expression of the enzyme (Stl 6 PDH) gene for controlling sorbitol synthesis in lactobacillus plantarum, a high-copy plasmid pIB184 is selected as an expression vector, recombinant plasmids are constructed, pIB184-srld1 and pIB184-srld2 over-expression plasmid maps are shown in figure 1, the obtained recombinant plasmids are electrically transferred into lactobacillus plantarum AR113 delta ldh competence for expression, and transformants are selected and named as AR113 delta ldh-srld1 and AR113 delta ldh-srld2. Recombinant plasmids were also transferred into wild-type AR113 competence as a control group to give strains AR113-srld1 and AR113-srld2 (FIG. 4 a). The Δldh strain was derived from a laboratory and verified in FIG. 4b, lanes 5-6 represent knockdown l-ldh and d-ldh verifies that the band size is 1000bp smaller than positive.
2.2 analysis of expression of target proteins and levels of digested sorbitol in recombinant strains
Biosynthesis of sorbitol in Lactobacillus plantarum begins first with fructose-6-phosphate, an intermediate of glycolysis, and then catalyzes the reduction of fructose 6-phosphate to sorbitol-6-phosphate via sorbitol-6P dehydrogenase (Stl 6 PDH) (EC: 1.1.1.140), and its reverse reaction, oxidation of sorbitol-6-phosphate. srlD is sorbitol-6-phosphate dehydrogenase derived from Lactobacillus plantarum, and is a key gene for sorbitol synthesis. Therefore, the expression of Stl6PDH encoded by srld gene was measured, and the change in sorbitol content was laterally reflected by the relative expression level of the gene.
Protein level detection of recombinant strains by SDS-PAGE, FIG. 5a shows that the supernatant fractions of recombinant strains AR113-srld2 and AR113 Deltaldh-srld 2 show a distinct protein band at about 25kDa, which is close to the target protein size of 28.8kDa, and the band gray scale of the size in the supernatant fractions of control groups AR113 and AR113 Deltaldh is significantly lower than that of the experimental group; the same size bands were observed in the crushed solution of each strain, and no significant differences occurred, indicating that sorbitol-6P dehydrogenase was expressed in the strain; and the expression of Stl6PDH protein was demonstrated by Western results, AR113-srld2 and AR113 Δldh-srld2, detected specific bands in the strain-disrupted supernatants (FIG. 5 b).
The expression level of srld in the different recombinant strains was further analyzed by qPCR (fig. 5c,5 d). The result shows that the expression level of srld in recombinant strain is improved by pIB184 plasmid, the expression level of srld in wild strain is improved by 500 times, and the expression level is improved to 3000 times after lactic acid is knocked out, and the result shows that the existence of lactic dehydrogenase can influence the expression of srld gene, and the expression level of srld can be obviously improved by over-expressing srld gene on the basis of lactic acid dehydrogenase is knocked out. In some cases increase aloneAdding expression levels or deleting individual genes to modulate intracellular activity is an inefficient process because single step flux control is often inefficient and requires additional metabolic manipulations. Furthermore, double knockout of l-ldh and d-ldh in Lactobacillus plantarum resulted in very low lactate synthesis, making the strain more dependent on Stl6PDH for NAD production + While sorbitol production is more dependent on the availability of NADH as a cofactor.
5.3 analysis of the freeze-drying survival of recombinant strains
In the wild type over-expressed strain, the over-expression of the srld gene does not significantly improve the survival rate of the strain under the condition of PBS, and the freeze-drying survival rate of the strain is not improved after the sorbitol protective agent is exogenously added, because the over-expression of the srld gene in the wild type strain alone does not catalyze the generation of sorbitol, the protective effect of the Stl6PDH on the strain in the freeze-drying process is lower than that of the wild type strain in the situation. In the strain with the ldh knocked out, the survival rate of the AR113 delta ldh-srld2 strain is obviously higher than that of the AR113 delta ldh and the AR113 delta ldh-srld1 under the PBS condition, and the freeze-drying survival rate of the strain reaches 41.94 percent, which is improved by 3.2 times compared with a control group. Consistent with the conditions under PBS, the lyophilization survival rate of both the added sorbitol and trehalose of AR113 Deltaldh-srld 2 was significantly higher than that of the other two strains, wherein the lyophilization survival rate under the promotion of trehalose addition was 74.3%. Meanwhile, the survival rate of the AR113 delta ldh-srld1 strain is also improved, but the improvement effect is not obviously different. Therefore, the srld gene is over-expressed on the basis of the ldh strain knocked out, enough cofactors are generated, the metabolic reaction of sorbitol is reversed, stl6PDH is promoted to progress to the direction of sorbitol production, and the survival effect of the strain after the freeze-drying process is superior to that of a control group in this case.
In conclusion, overexpression of srld gene alone in wild type only promotes Stl6PDH catabolism of sorbitol, but the effect of producing intracellular sorbitol is not achieved, so that the damage of the strain after freeze drying is higher than that of the wild type; the srld gene is overexpressed on the premise of knocking out ldh, the decomposition of sorbitol is reversed, the induced strain is carried out towards the direction of sorbitol production, and the damage of the AR113 delta ldh-srld2 strain after freeze drying is minimal. The sorbitol metabolism consumption way is reversed by the genetic engineering technology, so that the strain is carried out in the direction of sorbitol generation, the freezing resistance of the strain is improved, and the protection effect of the increase of the intracellular protectant on the freeze drying process of the strain is further demonstrated.
5.4 PEF-loaded sorbitol enters cells to improve freeze-drying survival rate of lactobacillus plantarum
PEF treatment sorbitol was loaded into four lactobacillus plantarum (AR 495, AR514, AR307 and WCFS 1), and freeze-drying survival of the strain was improved by sorbitol loaded into the cells. The freeze-drying survival rate of all strains under the condition of loading sorbitol in cells is improved compared with that of a control group, and the improvement times are as follows: AR495:2.3, AR514:5.5, AR307:2.45 and WCFS1:6.53. therefore, the method of loading the protective agent by PEF can improve the freezing resistance of the 4 strains of lactobacillus plantarum. The freeze-drying survival rate of AR514 under the condition of normal sorbitol addition is less than 10%, and the survival rate of the loaded sorbitol after entering cells is more than 33%, so that the survival rate is remarkably improved.
5.5 PEF-loaded sorbitol into cells to improve freeze-drying survival rate of probiotics
Effect of PEF-loaded sorbitol on different probiotics (lactobacillus plantarum AR113, lactobacillus thermophilus S-3 and lactococcus lactis NZ 9000) through the freeze-drying process: the freeze-drying survival rate of all three strains is improved after being treated by sorbitol medium PEF, wherein the freeze-drying survival rate of S-3 is improved from 10% to 55.56%, the freeze-drying survival rate of the three strains after being treated by sorbitol medium PEF NZ9000 is improved from 28% to 31%, and the freeze-drying survival rate of AR113 is improved from 24.1% to 71.6%. Thus loading sorbitol into the interior of the strain via PEF can improve the freeze-drying survival rate of the probiotic.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for improving the survival rate of a strain is characterized in that the ldh gene in lactobacillus plantarum is knocked out, and then the srlD gene is introduced into the lactobacillus plantarum, so that the sorbitol yield in the lactobacillus plantarum is increased, and the freeze-drying survival rate of the lactobacillus plantarum is improved;
the srlD genes comprise srlD1 genes and srlD2 genes,
the nucleotide sequence of the srld1 gene is shown as SEQ ID NO.12,
the nucleotide sequence of the srld2 gene is shown as SEQ ID NO. 13.
2. The method for improving survival rate of a strain according to claim 1, wherein the ldh gene comprises a l-ldh gene and a d-ldh gene, and the nucleotide sequence of the l-ldh gene is shown in SEQ ID NO. 11; the nucleotide sequence of the d-ldh gene is shown in SEQ ID NO. 10.
3. The method of claim 1, wherein the srlD gene is a sorbitol-6-phosphate dehydrogenase encoding gene of a sorbitol operon.
4. The method of claim 1, wherein the srlD gene is derived from WCFS1 strain.
5. The method of claim 1, wherein the lactobacillus plantarum is lactobacillus plantarum AR113.
6. The method for improving survival of a strain according to claim 1, wherein the sorbitol is produced by the following steps: the fructose-6P is catalyzed and reduced by sorbitol-6P dehydrogenase to generate sorbitol-6P, and the sorbitol-6P is oxidized to generate sorbitol.
7. The method of claim 6, wherein sorbitol production is dependent on NADH as a cofactor.
8. The method according to claim 7, wherein the ldh gene in lactobacillus plantarum is knocked out, the srlD gene is introduced into lactobacillus plantarum, and the srlD gene is overexpressed on the basis of knocking out the ldh strain to generate excessive NADH auxiliary factors, reverse the metabolic reaction of sorbitol and promote the sorbitol-6P dehydrogenase to proceed in the direction of sorbitol production.
9. The method of claim 8, wherein the lactobacillus plantarum strain harboring the srld gene grows normally.
10. The method for improving the survival rate of a strain according to claim 8, wherein after the ldh gene in the lactobacillus plantarum is knocked out, a lactobacillus plantarum mutant is obtained, the lactobacillus plantarum mutant generates excessive NADH for glycolytic conversion of an available carbon source, so that a metabolic pathway for oxidizing NADH is activated, a catabolic pathway for sorbitol utilization is reversed, the srlD gene is introduced into the lactobacillus plantarum, so that overproduction of sorbitol-6P dehydrogenase is realized, glycolytic flux is shifted from fructose-6P to final product sorbitol, and the generation of sorbitol in the lactobacillus plantarum is realized, so that the freeze-drying survival rate of the lactobacillus plantarum is improved.
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