Team:NWU-CHINA-B/Proof Of Concept

NWU-CHINA-B

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  • Conclusion

    •      A rare ginsenoside with high pharmacological activity was prepared by transforming ginsenoside Rb1 with high substrate concentration.For the purpose of CK, the production of SS-bgly gene from Sulfolobus solfataricus in pichia coli expression system and its transformation of ginsenoside were systematically studied, laying a foundation for the large-scale production of CK. The main conclusions are as follows:

        (1) The recombinant strain of Pichia pastoris GS115/9K-SS-bgly was successfully constructed. SS-bgly gene sequence was searched from NCBI database, and the recombinant expression vector pPIC9K-SS-bgly was successfully constructed after codon optimization. Then the recombinant expression vector was transferred into P. Pastoris GS115 competent cells. A mutant GS115/9K-SS-bgly6# with the highest enzyme activity was obtained, and its enzyme activity was up to 70 U/mL and protein content was 1.5 mg/mL. The successful expression of GS115/9K-SS-bgly 6# was confirmed by SDS-PAGE.

        (2) The recombinant expression vector pPICZαA-SS-bgly was successfully constructed using the constructed recombinant plasmid pPIC9K-SS-bgly as A template and the target gene was amplified by PCR. The recombinant strain GS115/9K-ZαA-SS-bgly4# was obtained by using the previously constructed strain as the host through secondary electrical transfer, combined with the resistance plate and shaking flask fermentation with different concentration gradient. The enzyme activity of GS115/9K-ZαA-SS-bgly4# was 152 U/mL, which was 117 % higher than that, and the protein content was 1.8 mg/mL. The gene copy number was determined to be 4 by fluorescence quantitative PCR.

        (3) The enzyme conditions of SS-bgly transformation of ginsenoside substrate to CK were optimized. Under the optimized enzymatic reaction conditions,9 mg/mL SS-bgly could completely transform 10 mg/mL and 20 mg/mL ginsenoside substrate to 3.79 mg/mL and 7.58 mg/mL CK within 12 h and 30 h, respectively. When the concentration of ginsenoside substrate reached 30 mg/mL, the yield of CK was 9.39 mg/mL after 48 h reaction, and the conversion rate reached 82.5%, which was higher than the CK yield reported in most literatures. In addition, the concentration of Rb1 in ginsenoside substrates is 15 mg/mL, higher than the concentration of Rb1 substrates reported in most current literatures, which is conducive to the realization of large-scale production of CK.



  • Innovation

    •     The high expression of SS-bgly gene from Sulfolobus solfataricus was realized in Pichia system after codon optimization, and the enzymatic reaction conditions of SS-bgly transformation of ginsenoside Rb1 to CK were explored. Under the optimized enzymatic reaction conditions, The recombinant SS-bgly can transform ginsenoside Rb1 with higher substrate concentration into CK, which is conducive to the large-scale production of CK.



  • ⅠConstruction of a recombinant strain expressing β -glycosidase in Pichia pastoris



    1. Construction of recombinant strain GS115-pPIC9K-SS-bgly

      2. After codon optimization of SS-bgly gene sequence, GC content decreased from 39.7% to 37.3%, and codon adaptation index (CAI) increased from 0.74 to 0.93. After the successful strain constructed by the company was activated, the pPIC9K SS bgly plasmid was extracted. After Sal Ⅰ enzyme was linearized, it was transferred to competent cells and coated on MD plate. For the single colony on the plate, the genome gene can be released by repeated freezing and thawing, and the supernatant can be taken for PCR verification. The results are shown in the figure. There is a target band at about 2000 bp, which is consistent with the theoretical size, while the blank control is GS115-pPIC9K has only a 500 bp band, which proves that the construction of the recombinant strain is successful.





      MD plate screening transformant





    2. Screening of high enzyme activity transformants



      3. Standard curve of pNP concentration and absorbance value




      The enzyme production activities of different transformants are also slightly different. In order to obtain transformants with high enzyme activity, 6 transformants successfully verified by colony PCR are selected for shake flask fermentation screening, and their enzyme activity is between 32-70 U / mL, of which 6# transformants have the highest enzyme activity, 70 U / ml.







      Enzyme activity of some transformants




    3. Growth curve, enzyme activity and protein content determination

      4. Activate GS115-pPIC9K-SS-bgly 6# transformant on YPD plate, ferment after single bacteria grow, take samples every 24 hours, dilute the sampled bacterial solution properly with ultrapure water, determine OD600, and then the supernatant was centrifuged to determine the β -glycosidase activity and protein content. The results of glycosidase activity and protein content are shown in the figure below. In the first 72 hours, the recombinant strain grew rapidly, then the growth rate slowed down and reached a stable level at 120 hours. The activity of β- glycosidase kept increasing in 0-120 h, reached the highest value of 70 U / mL at 120 h, and then decreased gradually. The protein content increased steadily and reached 1.5 mg / mL at 120 h. The protein content continued to increase after methanol induction, but the enzyme activity decreased. It is speculated that it may be caused by the accumulation of protease.







      Fermentation process curve







      Standard curve between standard protein concentration and absorbance value




    4. SDS-PAGE analysis

      5. After 120 h of methanol induction, 10 μL supernatant was taken for SDS-PAGE electrophoresis at the target molecular weight of 57 KDa.

        A clear band was observed nearby, indicating that SS-bgly gene was successfully expressed in P. pastoris.







      SDS-PAGE Electrophoretic figure




    This part mainly constructs the recombinant engineering strain gs115-ppic9k-ss-bgly Pichia pastoris. Firstly, the codon of SS -bgly gene was optimized and sent to the company for synthesis to obtain E.coli top 10 strain containing pPIC9K SS bgly plasmid. After the strain was activated, the plasmid was extracted, linearized by Sal I enzyme, and then transferred to Pichia pastoris GS115. The positive transformants were preliminarily screened by MD plate and colony PCR. After the successful transformants were screened by shake flask fermentation, a transformant with high-enzyme activity was obtained. During the fermentation process, the recombinant strain grew normally, After 120 hours of methanol induction, the enzyme activity and protein content can reach 70 U / mL and 1.5 mg / mL respectively. Take 10μL supernatant for SDS-PAGE detection, and the target band appears at 57 KDa, which proves that SS-bgly gene is successfully expressed in Pichia pastoris.







  • Ⅱ  Construction of recombinant strain with high expression of β -glycosidase



    1. Gene amplification

      2.   Using the plasmid pPIC9K-SS bgly extracted and preserved in the first part as the template, the enzyme digestion sites were amplified by primers.

        The SS-bgly gene fragments of Kpn Ⅰ and Not Ⅰ were about 1500 bp . The electrophoresis results showed that they were successfully amplified.





      Gel electrophoresis of SS-bgly gene amplification





    2. Verification of recombinant plasmid by double enzyme digestion

      3.   The two ends of the gene SS-bgly amplified by PCR were introduced into the enzyme digestion sites Kpn Ⅰ and not Ⅰ, and the expression vector pPICZ-α-A was digested by double enzymes, purified by electrophoresis, recovered, connected and transformed into competent cells of large intestine, and then evenly coated on low salt LB plate with corresponding resistance.The colony PCR results showed that the band size was 2000 bp, which was consistent with the theory. The positive clones verified by PCR were selected for further enzyme digestion verification. The results are shown in the figure. After double enzyme digestion,pPICZaA and SS-bgly bands with the same size as the theory appeared , combined with the sequencing results of the synthetic company, confirmed the plasmid pPICZαA-SS-bgly was successfully constructed.







      Verify positive inverters by colony PCR




      Identification of recombinant plasmid ppiczaa SS bgly by enzyme digestion




    3. Screening of positive transformants

      4. The plasmid pPICZαA-SS- bgly was extracted from the successfully sequenced strain after overnight culture, and Sac ⅰ enzyme line was used. After sexualization and purification, the strain GS115-pPIC9K-SS-bgly 6# selected in Chapter 2 was transferred to YPD. Sterile water was used to collect single colonies grown on resistant tablets (containing 100 g/mL Zeocin) and gradient coating was applied to YPD tablets with different resistance. The final screened inverters were released by repeated freezing and thawing operations, and PCR was performed with primers to verify the results, as shown in the figure. Obvious bands appeared at about 3000 bp, which was consistent with the theoretical value, indicating that the gene had been successfully inserted into the target strain.







      PCR identification of recombinant yeast strains




    4. Screening of high-activity strains

      5. In order to screen for high-activity, different inverters showed slightly different enzyme activity in shaker test. The transformants were selected through high resistance plate screening and colony PCR verification for shake-flask fermentation, and their enzymes. They lived in the range of 102-152 U/mL, with the highest activity of 152 U/mL in the 4# transforter.







      Invertor enzyme activity




    5. Comparison of fermentation characteristics between GS115/9K-SS-BGLY 6# and GS115/9K-Zα A-SS-BGLY 4#

      6. The recombinant strain GS115/9K-Zα A-SS-bgly 4# was fermented and the growth curve, enzyme activity and protein content were determined. As shown in the figure, there was no difference in the growth of recombinant GS115/9K-SS-bgly 6# and GS115/9K-Zα A-SS-bgly 4#. The enzyme activity and protein content of GS115/9K-Zα A-SS-bgly 4# were higher than that of GS115/9K-SS-bgly 6#. The enzyme activity of GS115/9K-SS-bgly 6# kept increasing from 0 to 120 h, then gradually decreased, and reached the highest value of 152 U/mL at 120 h, which increased by 117 %. The protein content has been increasing steadily, reaching 1.8 mg/mL after methanol induction for 120 h, a 20% increase compared with that of methanol induction. The protein content still increased after methanol induction, but the enzyme activity decreased, which may be due to the accumulation of protease, leading to the degradation of the target protein.








    6. The copy number of genes was determined by fluorescence quantitative PCR

      7. △CT=(CT, BGLY-CT,GAP)Test-(CT, bgly-ct (GAP)Calibrator calculated the mean CT values of target gene and reference gene and the expression level of target gene SS-bgly in transforant GS115/9K-SS-bgly 6# and transforant GS115/9K-ZαA-SS-bgly 4#. Using plasmid pPIC9KSS-bgly as single copy, the copy numbers of SS-bgly gene in GS115/9K-SS-bgly 6# and GS115-9K-Zα A-SS-bgly 4# were 1.89 and 4.03, respectively, and were round 2 and 4.







      Calculation of gene copy number




        Note 2: GAP was the internal reference gene and was represented by Ref; Plasmid pPIC9K-SS-bgly was used as reference sample and was represented by Cal. SS-bgly stands for Target gene and is represented by Target. Strains GS115-9K-bgly 6# and GS115-9K-Zα A-bgly 4# were used as Test samples.




    Conclusion

    The SS-bgly gene sequence was amplified from the plasmid pPIC9K-SS-bgly extracted from the first part as A template, and was linked to the vector pPICZαA. E. Coli TOP 10 strain containing pPICZα A-SS-bgly plasmid was successfully constructed. After the strain was activated on the low-salt LB resistance plate, the plasmid was extracted and linearized by restriction endonuclease Sac ⅰ enzyme. Then the plasmid was transferred to GS115-pPIC9K-SS - bgly 6# strain constructed in the first part. The transformants were preliminarily screened by resistance plate and colony PCR with different concentration gradients. The high activity strain GS115/9K-Zα A-SS- bgly 4# was obtained by shaking flask fermentation. After 120 h of methanol induction, its enzyme activity was up to 152 U/mL, which was increased by 117 %, and its protein content was 1.8 mg/mL, which was increased by 20 %. The gene copy number was 4 by fluorescence quantitative PCR.







  • Ⅲ  Optimization of transformation conditions of ginsenoside Rb1 with β -glycosidase



    1. Determination of temperature and pH of enzymatic reaction system

      2.   Under the condition that other conditions remain the same, the effect of SS-bgly on the activity of converting ginsenoside substrate to CK was studied in the range of 40-90 ℃. As shown in the figure, when the temperature was 40-80 ℃, the activity of SS-bgly converting ginsenoside substrate to CK increased with the increase of temperature. However, when the temperature increases to 90 ℃, the activity of CK production decreases. Therefore, 80 ℃ is the best reaction temperature for SS-bgly hydrolysis of ginsenoside substrate. The temperature of SS- bgly converting ginsenoside substrate to CK is higher than that of most ginsenoside hydrolases reported, and its high temperature resistance makes it more suitable for industrial transformation of ginsenoside substrate to CK. When the pH was 4.0-6.0, the activity of SS-bgly transforming ginsenoside substrate to produce CK increased with the increase of pH, while when the pH increased to 7.0, the activity of SS-bgly transforming ginsenoside substrate to produce CK decreased. Therefore, the optimum reaction pH for SS-bgly hydrolysis of ginsenoside substrate is 6.0.




        When the pH was 4.0-6.0, the activity of SS bgly transforming ginsenoside substrate to produce CK increased with the increase of pH, while when the pH increased to 7.0, the activity of SS bgly transforming ginsenoside substrate to produce CK decreased. Therefore, the optimum reaction pH for SS bgly hydrolysis of ginsenoside substrate is 6.0 .




    2. Effect of metal ions on saponin transformation

      3. The addition of ZnCl2, KCl, CaCl2 and FeCl3 metal ions in the enzyme reaction system had no effect on the activity of transforming ginsenoside substrate to produce CK, while the addition of CuCl2 metal ions reduced the activity of CK production to 57.2%. The addition of LiCl and MgSO4 metal ions can increase the activity of CK production by 25% and 15%, respectively. In order to determine the optimal type and concentration of metal ions, further experiments were designed. The results are shown in the figure. When LiCl and MgSO4 metal ions were added to the enzyme reaction system and the final concentration was 3 mm, their CK production activity was the highest. Under the optimal metal ion concentration, the activity of LiCl metal ions for CK production is 1.15 times that of MgSO4. Therefore, LiCl metal ions with a final concentration of 3 mM were added in the subsequent enzymatic reactions to promote the production of CK.
















    3. Determination of optimal substrate concentration

      4. When the temperature of the enzymatic reaction system is 80 ℃ and pH is 6.0, the final concentration of LiCl metal ions is 3 mM. The effect of substrate concentration on saponin transformation was investigated.







    4. Determination of optimal enzyme concentration

      5. The temperature of the enzymatic reaction system was set as 80 ℃, pH 6.0, the final concentration of LiCl metal ions was 3 mM, and the bottom was set as. The relationship between CK yield and enzyme concentration was studied when the concentration was 30 mg/mL.







      When the amount of enzyme increased from 0 mg/mL to 9 mg/mL, the yield of CK increased with the increase of the amount of enzyme; when the amount of enzyme increased to 12 mg/mL, the yield of CK increased more slowly than before. Therefore, 9 mg/mL was confirmed to be the optimal concentration of enzyme.







    5. Kinetic parameters

      6. The kinetic parameters are shown in the table. It can be seen that the values of Km, kcat and kcat / Km follow the order of RD > RB1 > F2, F2 > RB1 > RD and F2 > RB1 > Rd respectively, indicating that the catalytic efficiency of SS-bgly for ginsenoside F2 is much higher than RB1 and Rd, and F2 is immediately converted to CK once it appears.







    6. Transformation path analysis

      7. In the HPLC chromatogram, ginsenoside Rb1, Rd, F2 and CK standards showed obvious peaks at about 6 min, 14.5 min and 17 min respectively. It can be seen from the figure that SS-bgly transformed 5 mg / mL ginsenoside substrate (including 2.5 mg / ml Rb1 and 0.75 mg / mL Rd). After 3 hours, Rb1 completely disappeared and transformed into 1.52 mg / mL RD and 0.87 mg / ml CK. Until 6 hours after the reaction, Rd completely disappeared and transformed into CK. Therefore, the hydrolysis path of SS-bgly transforming ginsenoside substrate to CK is Rb1 → Rd → F2 → CK.













    Conclusion：

    The constructed strain was fermented, and the concentrated crude enzyme was obtained by salting out, dialysis and ultrafiltration. Enzymatic reaction was carried out in sodium acetate buffer. A series of optimization of enzymatic reaction conditions were carried out. It was found that when the temperature of enzymatic reaction system was 80 ℃, pH 6.0 and the final concentration of LiCl metal ions was 3 mM, 9 mg/mL SS-bgly can completely transform 10 mg/mL and 20 mg/mL ginsenoside substrates into 3.79 mg/mL and 7.58 mg/mL CK within 12 h and 30 h, respectively. When the substrate concentration reached 30 mg/mL, the yield of CK reached 9.39 mg/mL and the conversion rate reached 82.5% after 48 h of reaction, while when the substrate concentration was further increased to 40 mg/mL, the yield of CK decreased to 9.08 mg/mL and the conversion rate was 59.8%. The transformation pathway of ginsenoside Rb1 by HPLC was Rb1→ Rd→ F2→ CK.