A method for improving the yield of Nostoc polysaccharide by modifying PGD protein through point mutation, protein and application

Abstract

The present invention belongs to the technical field of industrial microorganisms, and discloses a PGD-C-I mutant protein modified by point mutation, wherein the amino acid sequence of the mutant protein is SEQ ID NO. 1. The method of the present invention replaces the amino acid residues that undergo glutathionylation modification in the PGD protein by heterologous expression and point mutation, thereby obtaining a PGD mutant protein with reduced enzyme activity, which is named PGD-C-I; the enzyme activity thereof is reduced relative to the original PGD protein, thereby increasing the yield of Nostoc polysaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial microorganisms, and in particular to a method for improving the yield of Nostoc mossula polysaccharide by transforming PGD protein through point mutation, the protein and application thereof. Background Art

[0002] Nostoc, also known as Nostoc hair-shaped cyanobacteria, is a first-class protected terrestrial cyanobacteria in my country. It is a prokaryotic microorganism that can perform photosynthesis. As a photoautotrophic microorganism, Nostoc can secrete extracellular polysaccharides during its growth, which can not only enhance the body's immunity, anti-tumor, anti-inflammatory and lower blood lipids, but also regulate intestinal flora, increase the proportion of beneficial bacteria, and improve the symptoms of pollen sensitivity, making Nostoc polysaccharides gradually widely used in the fields of biology, medicine, food, etc.

[0003] Microbial exopolysaccharides are renewable, biosafe, biodegradable, and biocompatible; and cyanobacteria have the advantages of autotrophic growth, rapid reproduction, strong adaptability, and large-scale cultivation. At present, the commercial production of most microbial exopolysaccharides is based on heterotrophic bacterial fermentation, while photoautotrophic cyanobacteria are a new source of natural polymers. In recent years, the research on the production of exopolysaccharides using cyanobacteria has received increasing attention.

[0004] However, the low yield of polysaccharides from Nostoc is the main factor restricting the large-scale production of its extracellular polysaccharides. At present, the main methods to improve the yield of microbial polysaccharides are to optimize the culture conditions and fermentation process, establish high-throughput screening or mutagenesis methods to select strains that can efficiently synthesize polysaccharides, and obtain strains with high polysaccharide yields through gene transduction or gene editing technology. For example, the Chinese invention patent CN116814441A, the patent name is: Application and method of gold nanomaterials in improving the yield of polysaccharides and triterpenes of Ganoderma lucidum active products. This invention can stably promote the synthesis of polysaccharides and triterpenes of Ganoderma lucidum active products by adding a certain amount of gold nanomaterials to its liquid fermentation medium. Chinese invention patent CN115747247A, the patent name is: A method, strain and application for rapid screening of high-yield β-glucan yeast strains. This invention verifies that there is a good linear relationship between the fluorescence intensity of fusion gene co-expression (GAS1-RFP gene sequence) and the yield of β-glucan. The expression of GAS1 gene increases, the fluorescence intensity increases, and the yield of glucan increases, building a bridge between fluorescence intensity and β-glucan yield. Chinese invention patent CN116445513A, the patent name is: Application of sll0528 gene in improving the extracellular polysaccharide production of Synechocystis PCC6803. This invention knocks out the sll0528 gene in Synechocystis PCC6803 by homologous recombination to obtain a strain of Synechocystis PCC6803 with improved extracellular polysaccharide production △sll0528. However, these technologies have the disadvantages of high cost, low efficiency and great difficulty, and are not applicable to improving the polysaccharide production of Nostoc. Through searching, no relevant patent public documents on regulating polysaccharide production from the perspective of protein glutathionylation modification have been found. In order to overcome the shortcomings of the prior art, the present invention increases the yield of Nostoc polysaccharide from the perspective of protein glutathionylation modification. Studies have shown that PGD protein is negatively correlated with the yield of Nostoc polysaccharide and the PGD protease activity increases after glutathionylation modification, which inhibits the synthesis of Nostoc polysaccharide. Therefore, the present invention uses point mutation technology to prevent modification and reduce enzyme activity, so as to increase the yield of Nostoc polysaccharide and promote industrial development and application. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method, protein and application for improving the yield of Nostoc mossula polysaccharide by modifying PGD protein through point mutation.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A PGD-CI mutant protein modified by point mutation, the amino acid sequence of the mutant protein is SEQ ID NO.1.

[0008] The method for constructing the PGD-CI mutant protein as described above comprises the following steps:

[0009] (1) Construction of PGD recombinant plasmid in Nostoc morifolia;

[0010] (2) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria;

[0011] (3) Digest the template plasmid DNA with Dpn I;

[0012] (4) PCR product self-circularization and transformation;

[0013] (5) Confirmation of mutant PGD-CI.

[0014] Furthermore, the specific steps are as follows:

[0015] (1) Extraction of Nostoc flagelliformis genome: Extraction was performed according to the instructions of the genome extraction kit (Vazyme, DC104-01);

[0016] (2) Acquisition of PGD gene fragments: Using the above-extracted genome as a DNA template, PGD-F and PGD-R as specific primers for the PGD gene, PCR amplification was performed according to the reaction system and procedure in Tables 1 and 2, and the amplified product was detected by agarose gel electrophoresis and then recovered;

[0017] PGD-F: SEQ ID NO.2:

[0018] TGCCGCGGCAGCCATAtgATGACACTACAAAGCTTTGGTGTGA

[0019] PGD-R: SEQ ID NO.3:

[0020] TGGTGGTGGTGGTGCTCGAGTTACTTCTTAGCTTCAGCAATGGGTACC

[0021] Table 1 PCR reaction system

[0022]

[0023]

[0024] Table 2 PCR amplification program

[0025]

[0026] (3) Linearization of E. coli pET-28a: The nucleic acid sequence of the vector pET-28a was provided by SnapGene. Upstream and downstream primers were designed according to the insertion site of the target gene in the vector:

[0027] F: SEQ ID NO.4: CTCGAGCACCACCACCACC;

[0028] R: SEQ ID NO.5: CATATGGCTGCCGCGCGG;

[0029] The PCR system and reaction procedure used were carried out according to step (2) Table 1 and Table 2, and the operation was carried out according to the template concentration of 30 ng / μL and the pre-denaturation time of 30 sec; the linearized plasmid was verified by agarose gel electrophoresis, and the correct fragment was purified and recovered;

[0030] (4) Ligation and transformation of PGD gene fragment with pET-28a linearized vector

[0031] (a) The purified and recovered PGD fragment was mixed with the vector according to the system in Table 3 below, and connected in a metal bath at 37° C. for 30 min to obtain a recombinant product;

[0032] Table 3 Ligation reaction system

[0033]

[0034] (b) 10 μL of the ligated product was added to the competent cell suspension and placed on ice for 30 min. This operation was performed in a clean bench.

[0035] (c) 42°C metal bath for 90 sec, ice bath for 2 min;

[0036] (d) Add 1 mL of antibiotic-free LB liquid medium and resuscitate in a shaking incubator at 37°C and 180 rpm for 1.5 h;

[0037] (e) The revived bacterial solution was centrifuged at 4500 rpm for 2 min, 900 μL of the supernatant was discarded, and the lower layer of bacteria was mixed by pipetting. 100 μL of the solution was spread on an LB solid plate containing 50 μg / ml kanamycin resistance and cultured in a 37°C incubator overnight;

[0038] (5) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria:

[0039] Design mutation primers according to mutation point:

[0040] PGD-CIF: SEQ ID NO.6: GGTATTATTATTCGCGCTCGCTTTTTGAATAAGA

[0041] PGD-CIR: SEQ ID NO.7: ACCTTTCCAAATCCGCGCCATTT

[0042] Perform PCR amplification according to the reaction system and procedures in Tables 1 and 2 of step (2). After the reaction is completed, take 5 μL of the PCR product for agarose gel electrophoresis to confirm the PCR product;

[0043] (6) Digestion of the template plasmid DNA with Dpn I: After the PCR reaction is completed, add Dpn I to the PCR reaction solution. Add 2 μL Dpn I to every 50 μL PCR reaction solution, mix gently, and react at 37°C for 1 hour.

[0044] (7) PCR product self-cyclization and transformation:

[0045] (a) Take out T4 Polynucleotide Kinase and Ligation high from the kit (Toyobo, SMK-101), melt them in an ice bath, and after melting, gently stir Ligation high to mix well;

[0046] (b) Take a new PCR tube and prepare the reaction solution as follows:

[0047] Table 4 PCR product self-cyclization system

[0048]

[0049] (c) Stir gently and react at 16°C for 1 hour;

[0050] (d) taking a portion of the reaction solution to transform Escherichia coli;

[0051] (8) Confirmation of mutant PGD-CI: Pick 4-8 ​​colonies, extract plasmids, and confirm by sequencing.

[0052] The use of the PGD-CI mutant protein as described above in increasing the yield of Nostoc morifolia polysaccharides.

[0053] A method for improving the yield of polysaccharides in Nostoc moss by modifying PGD protein through point mutation. The method comprises heterologously expressing the PGD protein in Nostoc moss, replacing the cysteine ​​residue at position 366 with isoleucine through point mutation to construct a PGD-CI mutant protein to reduce the protease activity of PGD, and transforming it into Nostoc moss by natural transformation, thereby improving the yield of polysaccharides in Nostoc moss.

[0054] The advantages and positive effects achieved by the present invention are:

[0055] 1. The method of the present invention replaces the amino acid residues that undergo glutathionylation modification in the PGD protein by heterologous expression and point mutation to obtain a PGD mutant protein with reduced enzyme activity, which is named PGD-CI; its enzyme activity is reduced compared with the original PGD protein, thereby increasing the yield of Nostoc polysaccharide.

[0056] 2. The method of the present invention is simple and reliable, easy to operate, suitable for large-scale promotion and application, has important theoretical and practical significance for further constructing high-yield polysaccharide genetic engineering bacteria, and has broad application prospects.

[0057] 3. The present invention mutates the PGD protein in Nostoc moss through heterologous expression and point mutation. The PGD protease activity is negatively correlated with the yield of Nostoc moss polysaccharide, and the enzyme activity of the PGD protein increases after glutathionylation modification. In order to reduce the PGD protease activity, the 366th cysteine ​​residue is replaced by isoleucine, thereby increasing the yield of Nostoc moss polysaccharide. The enzyme activity can be reduced by combining heterologous expression of Nostoc moss PGD protein with point mutation, and then it is transformed into Nostoc moss strain by natural transformation, thereby increasing the yield of Nostoc moss polysaccharide. The method of the present invention is simple, easy to operate, low-cost, and can be used to increase the yield of Nostoc moss polysaccharide. It has important theoretical and practical significance for further constructing high-yield polysaccharide genetic engineering bacteria, and has broad application prospects.

[0058] 4. PGD protein is negatively correlated with the yield of Nostoc polysaccharide, and the protease activity of PGD increases after glutathionylation modification. The present invention reduces the enzyme activity of PGD protein by point mutation of glutathionylation modified residues of PGD protein, thereby promoting the synthesis of Nostoc polysaccharide. The method of the present invention is simple, easy to operate, low-cost, and can be used to increase the yield of Nostoc polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a correlation diagram between PGD protease activity and Nostoc polysaccharide yield in the present invention;

[0060] Figure 2 This is a diagram showing the change in enzyme activity of the PGD protein before and after glutathionylation modification in the present invention;

[0061] Figure 3 It is the gel electrophoresis diagram of the PGD fragment in the present invention; wherein, M is DNA Marker;

[0062] Figure 4 It is the gel electrophoresis diagram of the linear amplification of the pET-28a vector in the present invention; wherein, M is DNA Marker;

[0063] Figure 5 It is the gel electrophoresis diagram of PGD-CI reverse PCR amplification in the present invention; wherein, M is DNA Marker;

[0064] Figure 6 Figure 2 is the sequencing result diagram of PGD-C-I DNA sequence (A) and protein sequence (B) in the present invention;

[0065] Figure 7This is a comparison diagram of the enzyme activity assay of the PGD protein before and after mutation in the present invention; wherein, WT: original PGD protein; PGD-CI: mutated protein;

[0066] Figure 8 This is a comparison chart of polysaccharide yield determination before and after PGD protein mutation in the present invention; wherein, WT: original strain of Nostoc mossoriasis; PGD-CI: Nostoc mossoriasis strain after PGD protein mutation. DETAILED DESCRIPTION

[0067] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0068] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0069] A PGD-CI mutant protein modified by point mutation, the amino acid sequence of the mutant protein is SEQ ID NO.1:

[0070] MTLQSFGVIGLAVMGENIALNVERNGFPIAVYNRSREKTDAFMAQRATGRNVKAAFTLEEFVALLERPRKILVMVQAGKPVDAVIAQLKPLLEEGDIIIDGGNSWFEDTERRTQELEPA GLRYLGMGVSGGEEGALNGPSLMPGGTTSSYEFLSPIFNKIAAQVDDGPCVTYIGPGGSGHYVKMVHNGIEYGDMQLIAEAYDLLKNVAGLDHNQLHEVFAEWNTTDELDSFLIEITKN IFPYIDPETKKPLVDLIVDAAGQKGTGRWTVQTALELGVAIPTITAAVNARIISSIKEERVAASKALTGPSGKYDGQTKDFINKVRDALYCSKICSYAQGMALLSTASKTYNWNLDLGE MARIWKGGIIIRARFLNKIKKAFSENPALPNLLLAPEFKQTILDRQTAWREVIATAATLGIPVPAFSASLDYFDSYRRDRLPQNLTQAQRDYFGAHTYLRLDKPGSFHTEWVPIAEAKK.

[0071] The method for constructing the PGD-CI mutant protein as described above comprises the following steps:

[0072] (1) Construction of PGD recombinant plasmid in Nostoc morifolia;

[0073] (2) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria;

[0074] (3) Digest the template plasmid DNA with Dpn I;

[0075] (4) PCR product self-circularization and transformation;

[0076] (5) Confirmation of mutant PGD-CI.

[0077] Furthermore, the specific steps are as follows:

[0078] (1) Extraction of Nostoc flagelliformis genome: Extraction was performed according to the instructions of the genome extraction kit (Vazyme, DC104-01);

[0079] (2) Acquisition of PGD gene fragments: Using the above-extracted genome as a DNA template, PGD-F and PGD-R as specific primers for the PGD gene, PCR amplification was performed according to the reaction system and procedure in Tables 1 and 2, and the amplified product was detected by agarose gel electrophoresis and then recovered;

[0080] PGD-F: SEQ ID NO.2:

[0081] TGCCGCGGCAGCCATAtgATGACACTACAAAGCTTTGGTGTGA

[0082] PGD-R: SEQ ID NO.3:

[0083] TGGTGGTGGTGGTGCTCGAGTTACTTCTTAGCTTCAGCAATGGGTACC

[0084] Table 1 PCR reaction system

[0085]

[0086]

[0087] Table 2 PCR amplification program

[0088]

[0089] (3) Linearization of E. coli pET-28a: The nucleic acid sequence of the vector pET-28a was provided by SnapGene. Upstream and downstream primers were designed according to the insertion site of the target gene in the vector:

[0090] F: SEQ ID NO.4: CTCGAGCACCACCACCACC;

[0091] R: SEQ ID NO.5: CATATGGCTGCCGCGCGG;

[0092] The PCR system and reaction procedure used were carried out according to step (2) Table 1 and Table 2, and the operation was carried out according to the template concentration of 30 ng / μL and the pre-denaturation time of 30 sec; the linearized plasmid was verified by agarose gel electrophoresis, and the correct fragment was purified and recovered;

[0093] (4) Ligation and transformation of PGD gene fragment with pET-28a linearized vector

[0094] (a) The purified and recovered PGD fragment was mixed with the vector according to the system in Table 3 below, and connected in a metal bath at 37° C. for 30 min to obtain a recombinant product;

[0095] Table 3 Ligation reaction system

[0096]

[0097] (b) 10 μL of the ligated product was added to the competent cell suspension and placed on ice for 30 min. This operation was performed in a clean bench.

[0098] (c) 42°C metal bath for 90 sec, ice bath for 2 min;

[0099] (d) Add 1 mL of antibiotic-free LB liquid medium and resuscitate in a shaking incubator at 37°C and 180 rpm for 1.5 h;

[0100] (e) The revived bacterial solution was centrifuged at 4500 rpm for 2 min, 900 μL of the supernatant was discarded, and the lower layer of bacteria was mixed by pipetting. 100 μL of the solution was spread on an LB solid plate containing 50 μg / ml kanamycin resistance and cultured in a 37°C incubator overnight;

[0101] (5) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria:

[0102] Design mutation primers according to mutation point:

[0103] PGD-CIF: SEQ ID NO.6: GGTATTATTATTCGCGCTCGCTTTTTGAATAAGA

[0104] PGD-CIR: SEQ ID NO.7: ACCTTTCCAAATCCGCGCCATTT

[0105] Perform PCR amplification according to the reaction system and procedures in Tables 1 and 2 of step (2). After the reaction is completed, take 5 μL of the PCR product for agarose gel electrophoresis to confirm the PCR product;

[0106] (6) Digestion of the template plasmid DNA with Dpn I: After the PCR reaction is completed, add Dpn I to the PCR reaction solution. Add 2 μL Dpn I to every 50 μL PCR reaction solution, mix gently, and react at 37°C for 1 hour.

[0107] (7) PCR product self-cyclization and transformation:

[0108] (a) Take out T4 Polynucleotide Kinase and Ligation high from the kit (Toyobo, SMK-101), melt them in an ice bath, and after melting, gently stir Ligation high to mix well;

[0109] (b) Take a new PCR tube and prepare the reaction solution as follows:

[0110] Table 4 PCR product self-cyclization system

[0111]

[0112] (c) Stir gently and react at 16°C for 1 hour;

[0113] (d) taking a portion of the reaction solution to transform Escherichia coli;

[0114] (8) Confirmation of mutant PGD-CI: Pick 4-8 ​​colonies, extract plasmids, and confirm by sequencing.

[0115] The use of the PGD-CI mutant protein as described above in increasing the yield of Nostoc morifolia polysaccharides.

[0116] A method for improving the yield of polysaccharides in Nostoc moss by modifying PGD protein through point mutation. The method comprises heterologously expressing the PGD protein in Nostoc moss, replacing the cysteine ​​residue at position 366 with isoleucine through point mutation to construct a PGD-CI mutant protein to reduce the protease activity of PGD, and transforming it into Nostoc moss by natural transformation, thereby improving the yield of polysaccharides in Nostoc moss.

[0117] Specifically, the relevant preparation and detection are as follows:

[0118] Example 1: Construction and expression of mutant recombinant protein, determination of enzyme activity and acquisition of engineered strains

[0119] 1. Correlation analysis between PGD protease activity and Nostoc polysaccharide yield and determination of PGD protein activity before and after glutathionylation

[0120] 1. Correlation analysis between PGD protease activity and Nostoc polysaccharide yield:

[0121] The yield of Nostoc polysaccharide was determined by the phenol-sulfuric acid method. The PGD protease activity was determined according to the instructions of the kit (Keming, 6PGDH-1-W). The yield of Nostoc polysaccharide and related enzyme activities under H2O2 induction were determined. Origin20.0 was used to analyze whether there was a correlation between the two. The results are as follows Figure 1 As shown in the figure, it can be seen that the PGD protease activity is negatively correlated with the yield of Nostoc polysaccharide.

[0122] 2. Determination of enzyme activity of PGD protein before and after glutathionylation modification:

[0123] The PGD protein was modified with different concentrations of H2O2, and then the enzyme activity before and after modification was measured according to the instructions of the kit (Keming, 6PGDH-1-W). The results are shown in Figure 2 As shown, by comparison, it was found that the protease activity of the modified PGD was enhanced.

[0124] 2. Construction of E. coli recombinant plasmid containing point mutations

[0125] 1. Extraction of Nostoc genome:

[0126] (1) The Nostoc moss cells cultured to an absorbance value of about 0.8 at 750 nm were examined under a microscope. The Nostoc moss cells in a chain state were selected for genome extraction.

[0127] (2) Take 10 mL of the Nostoc moss cell suspension, centrifuge and discard the supernatant, wash twice with fresh culture medium, transfer the cell pellet to a mortar pre-cooled with liquid nitrogen, quench with liquid nitrogen and grind for about 20 min. During the grinding process, avoid repeated freezing and thawing of the cells.

[0128] (3) The ground Nostoc moss powder was transferred to a pre-cooled EP tube and extracted according to the instructions of the genome extraction kit (Vazyme, DC104-01).

[0129] 2. Acquisition of PGD gene fragments:

[0130] The above-extracted genome was used as a DNA template, PGD-F and PGD-R were used as specific primers for the PGD gene, and PCR amplification was performed according to the reaction system and procedures in Tables 1 and 2. The amplified product was detected by agarose gel electrophoresis and then recovered. The results of agarose gel electrophoresis were as follows: Figure 3 As shown, the results show that the bands are clear and bright, with the correct size, and can be used for subsequent experiments.

[0131] PGD-F: TGCCGCGCGGCAGCCATAtgATGACACTACAAAGCTTTGGTGTGA

[0132] PGD-R:TGGTGGTGGTGGTGCTCGAGTTACTTCTTAGCTTCAGCAATGGGTACC

[0133] Table 1 PCR reaction system

[0134]

[0135] Table 2 PCR amplification program

[0136]

[0137] 3. Linearization of E. coli pET-28a:

[0138] Linearized plasmid: The nucleic acid sequence of the vector pET-28a was provided by SnapGene. Upstream and downstream primers were designed according to the insertion site of the target gene in the vector:

[0139] F:CTCGAGCACCACCACCACC R:CATATGGCTGCCGCGCGG

[0140] The PCR system and reaction procedure used were as shown in Tables 1 and 2. In addition, the template concentration was 30 ng / μL and the pre-denaturation time was 30 sec. The linearized plasmid was verified by agarose gel electrophoresis, and the correct fragment was purified and recovered. The results of agarose gel electrophoresis are shown in Figure 4 As shown, the results show that the bands are clear and bright, with the correct size, and can be used for subsequent experiments.

[0141] 4. Connect and transform the PGD gene fragment to the pET-28a linearized vector:

[0142] (1) The purified and recovered PGD fragment was mixed with the vector according to the system in Table 3, and connected in a metal bath at 37°C for 30 minutes to obtain a recombinant product.

[0143] (2) Add 10 μL of the ligated product to the competent cell suspension and place on ice for 30 min. This operation should be performed in a clean bench.

[0144] (3) 42℃ metal bath for 90 seconds, ice bath for 2 minutes.

[0145] (4) Add 1 mL of antibiotic-free LB liquid culture medium and revive the cells in a shaking incubator at 37°C and 180 rpm for 1.5 h.

[0146] (5) The revived bacterial solution was centrifuged at 4500 rpm for 2 min. 900 μL of the supernatant was discarded and the lower layer of bacteria was mixed by pipetting. 100 μL of the solution was spread on an LB solid plate containing 50 μg / ml kanamycin resistance and cultured in a 37°C incubator overnight.

[0147] Table 3 Ligation reaction system

[0148]

[0149] 5. Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria:

[0150] Design mutation primers according to mutation point:

[0151] PGD-CIF: GGTATTATTATTCGCGCTCGCTTTTTGAATAAGA

[0152] PGD-CIR:ACCTTTCCAAATCCGCGCCATTT

[0153] PCR amplification was performed according to the reaction system and procedure in Tables 1 and 2. After the reaction, 5 μL of PCR product was taken for agarose gel electrophoresis to confirm the PCR product. The results of agarose gel electrophoresis were as follows: Figure 5 As shown, the results show that the bands are clear and bright, with the correct size, and can be used for subsequent experiments.

[0154] 6. Digest the template plasmid DNA with Dpn I:

[0155] After the PCR reaction was completed, 2 μL of Dpn I was added to the PCR reaction solution (total volume 50 μL), mixed gently, and reacted at 37° C. for 1 hour.

[0156] 7. PCR product self-cyclization and transformation:

[0157] (1) Take out T4 Polynucleotide Kinase and Ligation high from the kit (Toyobo, SMK-101) and thaw them in an ice bath. After thawing, gently stir Ligation high to mix well.

[0158] (2) Take a new PCR tube and prepare the reaction solution as follows:

[0159] Table 4 PCR product self-cyclization system

[0160]

[0161] (3) Stir gently and allow to react at 16°C for 1 hour.

[0162] (4) Take a portion of the reaction solution to transform Escherichia coli.

[0163] 8. Confirmation of mutant PGD-CI:

[0164] Pick 4-8 ​​colonies, extract plasmids, and confirm by sequencing. The sequencing results are as follows: Figure 6 As shown, the sequencing results were correct and the mutant PGD-CI was successfully constructed.

[0165] 3. Extraction and purification of PGD protein and PGD-CI mutant protein

[0166] (1) The correctly sequenced recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. After overnight culture, a single colony was picked and inoculated into a 5 mL LB tube containing 50 μg / ml kanamycin resistance and cultured overnight at 37°C and 180 rpm in a shaking incubator.

[0167] (2) Inoculate the overnight culture into 100 mL of LB liquid culture medium containing 50 μg / ml kanamycin resistance at a volume ratio of 1:50, and expand the culture on a shaker. Measure the OD after about 1.5 h. 600 The cultivation ends when the value is around 0.8.

[0168] (3) Isopropylthiogalactoside (IPTG) was added at a final concentration of 0.5 mmol / L and protein expression was induced at 20°C for 20 h.

[0169] (4) The bacterial precipitate was collected by centrifugation at 8000 rpm for 15 min at 4°C, and the bacterial cells were suspended in lysis buffer. According to the total volume of the buffer, 1% by volume of lysozyme and 1% by volume of the protein inhibitor PMSF were added.

[0170] (5) The cells were disrupted by ultrasonic wave in an ice bath for 25 min. The disrupted bacterial solution was centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was the crude protein after expression.

[0171] (6) Use Ni + -NTA resin chromatography column was used to purify PGD protein and PGD-CI mutant protein.

[0172] IV. Enzyme activity assay of PGD protein and PGD-CI mutant protein

[0173] PGD ​​catalyzes the reaction of 6-phosphogluconate and NADP + Generate NADPH, which has a characteristic absorption peak at 340nm, and NADP +No; PGD activity was calculated by measuring the rate of increase of absorbance at 340 nm. The specific steps were determined according to the instructions of the kit (Ke Ming, 6PGDH-1-W). The results are shown in Figure 7 As shown, it can be seen that the activity of the PGD-CI mutant protein is reduced by 82.02% compared with the activity of the original PGD protein, thereby increasing the content of Nostoc moss polysaccharides.

[0174] 5. Transformation of PGD-CI mutant protein into Nostoc moss

[0175] The PGD-CI mutant protein gene was transformed into the Nostoc strain by natural transformation to obtain an engineered strain. The polysaccharide yield was determined by the phenol-sulfuric acid method. Figure 8 As shown in the figure, it can be seen that the polysaccharide yield is increased by 11.3% compared with the original strain. This result is very significant in increasing the polysaccharide yield of Nostoc morifolia.

[0176] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A PGD-CI mutant protein modified by point mutation, characterized in that: The amino acid sequence of the mutant protein is SEQ ID NO.

1.

2. The method for constructing the PGD-CI mutant protein according to claim 1, characterized in that: The steps include: (1) Construction of PGD recombinant plasmid in Nostoc morifolia; (2) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria; (3) Digest the template plasmid DNA with Dpn I; (4) PCR product self-circularization and transformation; (5) Confirmation of mutant PGD-CI.

3. The construction method according to claim 2, characterized in that: The specific steps are as follows: (1) Extraction of Nostoc flagelliformis genome: Extraction was performed according to the instructions of the genome extraction kit; (2) Obtaining PGD gene fragments: Using the above-extracted genome as a DNA template and PGD-F and PGD-R as specific primers for the PGD gene, PCR amplification was performed according to the following reaction system and procedure, and the amplified product was recovered after detection by agarose gel electrophoresis; PGD-F: SEQ ID NO.2; PGD-R: SEQ ID NO. 3; PCR reaction system: PCR amplification procedure: (3) Linearization of E. coli pET-28a: Design upstream and downstream primers according to the insertion site of the target gene in the vector: F: SEQ ID NO.4; R: SEQ ID NO.5; The PCR system and reaction procedure used were carried out according to step (2), and in addition, the operation was carried out according to the template concentration of 30 ng / μL and the pre-denaturation time of 30 sec; the linearized plasmid was verified by agarose gel electrophoresis, and the correct fragment was purified and recovered; (4) Ligation and transformation of PGD gene fragment with pET-28a linearized vector (a) The purified and recovered PGD fragment was mixed with the vector according to the following system, and connected in a metal bath at 37°C for 30 minutes to obtain a recombinant product; Ligation reaction system: (b) 10 μL of the ligated product was added to the competent cell suspension and placed on ice for 30 min. This operation was performed in a clean bench. (c) 42°C metal bath for 90 sec, ice bath for 2 min; (d) Add 1 mL of antibiotic-free LB liquid medium and resuscitate in a shaking incubator at 37°C and 180 rpm for 1.5 h; (e) The revived bacterial solution was centrifuged at 4500 rpm for 2 min, 900 μL of the supernatant was discarded, and the lower layer of bacteria was mixed by pipetting. 100 μL of the solution was spread on an LB solid plate containing 50 μg / ml kanamycin resistance and cultured in a 37°C incubator overnight; (5) Reverse PCR of PGD recombinant plasmid to obtain the template plasmid of PGD-CI mutant bacteria: Design mutation primers according to mutation point: PGD-CIF: SEQ ID NO.6; PGD-CIR: SEQ ID NO.7; Perform PCR amplification according to the reaction system and procedure of step (2). After the reaction is completed, take 5 μL of the PCR product and perform agarose gel electrophoresis to confirm the PCR product; (6) Digestion of the template plasmid DNA with Dpn I: After the PCR reaction is completed, add Dpn I to the PCR reaction solution. Add 2 μL Dpn I to every 50 μL PCR reaction solution, mix gently, and react at 37°C for 1 hour. (7) PCR product self-cyclization and transformation: (a) Take out T4 Polynucleotide Kinase and Ligation high from the kit, thaw them in an ice bath, and after thawing, gently stir Ligation high to mix well; (b) Take a new PCR tube and prepare the reaction solution as follows: PCR product self-cyclization system: (c) Stir gently and react at 16°C for 1 hour; (d) taking a portion of the reaction solution to transform Escherichia coli; (8) Confirmation of mutant PGD-CI: Pick 4-8 ​​colonies, extract plasmids, and confirm by sequencing.

4. Use of the PGD-CI mutant protein as claimed in claim 1 in increasing the yield of Nostoc flagelliformis polysaccharides.

5. A method for increasing the yield of Nostoc mossula polysaccharide by modifying PGD protein through point mutation, characterized in that: The method comprises heterologously expressing the PGD protein in Nostoc moss, replacing the 366th cysteine ​​residue with isoleucine through point mutation to construct a PGD-CI mutant protein to reduce the PGD protease activity, and transforming it into Nostoc moss by natural transformation, thereby increasing the yield of Nostoc moss polysaccharides; Wherein, the amino acid sequence of the PGD-CI mutant protein is SEQ ID NO.1.

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