D-psicose-3-epimerase (DAE) plays an important role in the biotransformation of D-psicose, but the thermal stability of D-psicose-3-epimerase is poor and the expression level is low, which cannot meet the requirements of industrial production. In this study, we choosed four origin of DAE, is respectively Thermoclostridium caenicola(TcDAE)，Novibacillus thermophilus(NtDAE), Thermogutta terrifontis(TtDAE), and Dorea sp. CAG 317(DsDAE). Then we ligated the coding genes into the expression vectors, and transformed them into Pichia pastoris GS115 strain. The target protein was induced and purified, and tested the yield of D-psicose (Figure 1). Based on the yield of D-alloulose, it can be screened which source of DAE has a higher yield and higher thermal stability.
On this basis, the liquid PTVA method was used to obtain high-copy strains by Zeocin antibiotic screening to further improve the expression level of D-psicose-3-epimerase. This study will provide a new strategy for the industrial production of D-psicose.
Figure 1.The engineering schematic diagram of the project design
1.Plasmid Construction
1.1 PCR amplification(target gene)
At present, we choose four origin of DAEs, is respectively Thermoclostridium caenicola(TcDAE)，Novibacillus thermophilus(NtDAE), Thermogutta terrifontis(TtDAE), and Dorea sp.CAG 317(DsDAE). The DNA encoding TcDAE,TtDAE,NtDAE and DsDAE were optimised according to the codon preference of E.coli and synthesised. To construct the expression plasmid for TcDAE,TtDAE,NtDAE and DsDAE, firstly, the gene encoding TcDAE,TtDAE,NtDAE and DsDAE was amplified using polymerase chain reaction (PCR) with primer. The obtained PCR product was digested with EcoRI and SalI and inserted into pPICZαA plasmids.
The results are showed in figure 2, all 4 target genes(TcDAE,TtDAE,NtDAE and DsDAE) are highlighted between 750 and 1000bp markers, which accurately matched with theoretical length and indicate the succeed of amplification.
Figure 2. The electrophoresis verification of the target fragment TcDAE,TtDAE,NtDAE and DsDAE. A.TtDAE: 873 bp B.NtDAE: 855bp C.TcDAE:870 bp D.DsDAE: 870 bp
1.2:Double Digestion(plasmid)
The plasmid pPICZαA was digested with EcoRI and SalI, and used the unoperated pPICZaA as control group.The electrophoresis verify the results of enzyme digestion, the results are shown in Figure 3. The double digestion pPICZαA are highlighted between 3000 and 5000bp, and the length is greater than the control group to verify the digestion was very successful.But the control group are already became a super-helical structure, so the electrophoretic migration speed is fast, so it is less than the actual length. turn to liner from curcle.
Figure 3 . The electrophoresis verification of digestion vector pPICZαA Note: 1)pPICZαA: 3593 bp 2)Double Digestion pPICZαA:3487bp
1.3:The Single Clone Verification and Sequencing Comparison(E.coli DH5α )
We transferred the plasmid pPICZαA-TcDAE into E.coli-DH5α and did single clone verification. Figure 4-A shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of the target gene. Figure 4-B, showed the E. coli plates and the location of sample for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 4-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-TcDAE.
Figure 4. The single clone verification and sequencing comparison of plasmid pPICZαA-TcDAE ( E.coli DH5 )
Figure 5. The single clone verification and sequencing comparison of plasmid pPICZαA-TtDAE ( E.coli DH5 )
The results plasmid pPICZαA-TtDAE(Figure 5), pPICZαA-NtDAE (Figure 6), and pPICZαA-DsDAE (Figure 7) are consistent with pPICZαA-TcDAE, which proves that the plasmid is also successfully constructed.
Figure 6. The single clone verification and sequencing comparison of plasmid pPICZαA-NtDAE ( E.coli DH5 )
Figure 7. The single clone verification and sequencing comparison of plasmid pPICZαA-DsDAE ( E.coli DH5 )
2.Protein expression
2.1:The transformed plate colony diagram(Pichia Pastories GS-115)
The recombinant plasmids were transformed into Pichia Pastoris GS115 for expression. Each kind of plasmid was cultured in three separate petri dishes. All 12 plate successfully developed colonies in Figure 8, which verified the plasmid (pPICZαA-TtDAE, pPICZαA-NtDAE, pPICZαA-DsDAE and pPICZαA-TcDAE) have successfully transformed into Pichia Pastories GS115.
Figure 8. The transformed plate colony diagram of pPICZαA-TtDAE,pPICZαA-TcDAE,pPICZαA-NtDAE and pPICZαA-TcDAE(Pichia pastoris GS115)
2.2:The colony PCR identification(Pichia Pastories GS115)
A single colony containing the recombinant plasmid was cultured in medium containing antibiotics (100 mg/mL Zeocin) at 30oC.Next, verify the transformation status using monoclonal antibodies.The gene represents a clear band falling between 750 and 1500 bp, which corresponds to the length of the target gene in the Figure 9.The result indicates successful transformation of Pichia Pastories GS115.
Figure 9. The colony PCR identification of pPICZαA-TtDAE/pPICZαA-TcDAE/pPICZαA-NtDAE/pPICZαA-TcDAE(Pichia pastoris GS115)
2.3: SDA-PAGE detection
When the cultures reached optical density (OD600) about 0.6-0.8, methanol was added to the BMMY for induction of the recombinant protein. After the induction,we induced sampling at 24 and 98 hours the bacterial cells were lysed by sonication in phosphate buffer.The recombinant His6-fused TtDAE,TcDAE,NtDAE and TcDAE were purified by Ni2+ at finity chromatography.The protein was detected by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
In both figure 10 A and B, there’s protein falling between 34kDa to 43kDa. The coherence between target protein size and the observed bands indicates successful protein expression in the 24h.
Figure10. The SDA-PAGE detection of the productsin the24h. A.SDA-PAGE detection of crude protein extract. B Purification of the recombinant His6-tagged protein Note: TcDAE:37.8kDa, TtDAE:37.3kDa, NtDAE:37.9kDa, DsDAE:37.8kDa
Next, the same steps were taken to process the 96 hour protein sample. In both figure 11A , the purified protein protein falling between 34 kDa to 43 kDa. The coherence between target protein size and the observed bands indicates successful protein expression in the 96h.
The expression level of proteins can be characterized by intden values. The bands of the target protein was cutted , and used ImageJ software to calculate the intden values of the protein bands (Figure 11B). The intden values were established by GraphPad Prism with five gray values in each group. The abscissa was TcDAE, TtDAE, NtDAE,DsDAE and CK(GS115), and the ordinate was intden value of protein. In Figure 11C, the intden value of NtDAE was significantly higher than that of CK (GS115) TcDAE, TtCAE, and DsDAE. It indicates that the protein expression level of NtDAE is higher than that of other groups.The protein expression of TcDAE and TtCAE was similar. The intden value of DsDAE compared with TcDAE and TtDAE, and the protein expression level of DsDAE is the lowest. But the intden value of DsDAE,TcDAE,TtDAE and NtDAE significantly higher than those in control group(GS115). It shows that the protein expression of DsDAE,TcDAE,TtDAE and NtDAE is successful.
Figure 11. The protein expression of TcDAE, TtSAE, NtDAE, and DsDAE at 96 hours. A represents the SDS-PAGE gel image of the purified protein after 96 hours of induction. B represents the intden value analysis image of Image J. C represents the intden value of different proteins, GS115 represents Pichia pastoris GS115.
3.Test
3.1 Production of D-psicose
Using 10mg/ml D-fructose as the substrate,we added 0.3 μmol of purified recombinase TcDAE,TtDAE,NtDAE,DsDAEand CK-GS115, 1 mmolL CoCl2.Then each recombinant DAE was reacted at 40,50,60,70 and80 degrees for 10min, and boiled for 10 min. After the reaction, the product was centrifuged and diluted to a certain concentration, and the content of D-psicose was detected by HPLC.
We used High performance liquid chromatography(HPLC) to detect the content of D-psicose by of weipu biological company, and the confidence interval was 0.05 %. HPLC detection conditions: fixed phase is 2695 HPLC Waters Sugar-PakI sugar column, and mobile phase is ultrapure water, The flow rate is 0.4 m /min, and column temperature is 85 °C.
Table 1 shows the detection data of D-psicose, which includes the average of three replicates for each sample group. The results indicate that D-psicose production was not detected in the blank control group and negative control group(Pichia pastoris GS115), while TcDAE, TtAE, NtDAE, and DsDAE proteins can catalyze D-fructose to generate D-psicos.
Table 1. Production of D-psicose at different temperatures
| Temperature/D-psicose production | 40° | 50° | 60° | 70° | 80° |
| Blank control | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| Pichia pastoris GS115 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| TcDAE | 1.988 | 2.038 | 2.144 | 0.829 | 0.000 |
| TtDAE | 1.174 | 2.666 | 2.317 | 0.017 | 1.585 |
| NtDAE | 2.557 | 2.656 | 2.860 | 3.470 | 2.608 |
| DsDAE | 1.108 | 1.365 | 1.930 | 2.017 | 1.165 |
Next, the production of D-psicose was analyzed using Graphpad software. The X axis represents temperature, and the Y axis represents the yield of D-psicose. The production of D-psicose, increasing with temperature, shows a trend of first increasing and then decreasing in figure 12A.Moreover, the TcDAE and TtDAE groups at 60 degrees showed the highest production of D-psicose, which indicating the enzyme activity of TcDAE and TtDAE are highest at 60 degrees.
In figure 12B, the production of D-alloulose in the NtDAE and DsDAE groups showed a trend of first increasing and then decreasing. The NtDAE and DsDAE groups at 70 degrees had the highest D-psicose production. It indicating that NtDAE and DsDAE proteins are active and have the highest activity at 70 degrees.
Figure 12. Line graph of D-psicose production at different temperatures. A represents the D-psicose production in the TcDAE and TtSAE groups. B indicate the D-psicose production of Nt DAE and TcDAE groups.
Comparing the D-psicose production of TcDAE, TtAE, NtDAE, and DsDAE groups, it was found that the D-psicose productivity of the NtDAE group was significantly higher than that of the TcDAE, TtAE, and DsDAE groups(Figure 13). There was no significant difference in the maximum yield among the TcDAE, TtAE, and DsDAE groups. Therefore, the NtDAE group has the highest yield and the highest thermal stability
Figure 13. Production of D-psicose at different temperatures
3.2 Conversion rate of D-psicose
3.2.1 Conversion rate of D-psicose in the TcDAE, TtAE, NtDAE, and DsDAE
Using D-fructose as a substrate, TcDAE, TtDAE, NtDAE, and DsDAE proteins were added to measure the yield of D-psicose, and the conversion rate of DAE could be calculated. Table 2 shows that the conversion rates of TcDAE, TtDAE, NtDAE, and DsDAE groups are 21.4%, 23.2%, 34.7%, and 20.2%, respectively.
Table 2. Maximum conversion rate of D-alloulose in TcDAE, TtAE, NtDAE,and DsDAE group
| Group | D-Fructose(mg) | D-psicose(mg) | Maximum Conversion rate(100%) |
| Blank control | 10 | 0 | 0% |
| Pichia pastoris GS115 | 10 | 0 | 0% |
| TcDAE | 10 | 2.144 | 21.4% |
| TtDAE | 10 | 2.317 | 23.2% |
| NtDAE | 10 | 3.470 | 34.7% |
| DsDAE | 10 | 2.017 | 20.2% |
3.2.2. PTVA method for screening high copy NtDAE and D-psicose production
Inoculate the expression strain of D-alloulose-3-isomerase from Novibacillus thermophilus into 3mL YPD medium with an initial concentration of Zeocin 500 μg/mL. Next, we centrifuge every 2 days, remove the supernatant, and inoculate in YPD medium with higher concentrations of antibiotics. The concentration gradients of Zeocin were 1 mg/ml, 2 mg/ml, 3 mg/ml, and 4 mg/ml, respectively. Finally, they were inoculated onto a 4mg/ml Zeocin YPD medium, and the yield of D-alloulose was measured using the same method by HPLC.
In figure 14 A, the growth of NtDAE group at various concentrations of Zeocin showed that there were more colonies at 500 μg/mL, and the number of colonies decreased with increasing concentration.
NtDAE-op stands for selecting high-yield NtDAE. In figure 14B, the D-psicose production of NtDAE group is lower than that of NtDAE-op group. And the table in figure 14B shows that the conversion rate of NtDAE group is 34.7%, and the conversion rate of NtDAE op group is 37.0%.
Figure 14. PTVA method for screening high copy NtDAE and D-psicose production