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     <h4>3.PTVA Method for Screening High Copy NtDAE and D-psicose Production</h4>
 
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         Inoculate the expression strain of D-allulose-3-isomerase from <em>Novibacillus thermophilus</em> into 3 mL YPD medium with an initial concentration of Zeocin 500 μg/mL. Next, 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 4 mg/mL Zeocin YPD medium, and the yield of D-allulose was measured using the same method by HPLC.
 
         Inoculate the expression strain of D-allulose-3-isomerase from <em>Novibacillus thermophilus</em> into 3 mL YPD medium with an initial concentration of Zeocin 500 μg/mL. Next, 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 4 mg/mL Zeocin YPD medium, and the yield of D-allulose was measured using the same method by HPLC.
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         <img src="https://static.igem.wiki/teams/5528/bba-k5528007/10.png" width="60%" alt="Figure 10: PTVA method for screening high copy NtDAE and D-psicose production">
 
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             <caption>Figure 10. PTVA method for screening high copy NtDAE and D-psicose production.</caption>
 
             <caption>Figure 10. PTVA method for screening high copy NtDAE and D-psicose production.</caption>

Revision as of 05:18, 29 September 2024


pPICZαA-NtDAE

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2282
    Illegal BamHI site found at 365
    Illegal XhoI site found at 3465
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1123
    Illegal NgoMIV site found at 1184
    Illegal AgeI site found at 84
  • 1000
    COMPATIBLE WITH RFC[1000]

<!DOCTYPE html> pPICZαA-NtDAE Project

Engineering Principle

D-fructose can be converted into D-psicose by D-psicose 3-epimerase (DAE) under the catalysis of CoCl2. In this study, we synthesized the coding genes of D-psicose-3-epimerase from different sources, ligated the coding genes into the expression vectors, and transformed them into Pichia pastoris GS115 strain. The target protein was induced and purified, and the yield and enzymatic properties of D-psicose and D-psicose-3-epimerase with good thermal stability were detected. 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 and the yield of D-allulose. This study will provide a new strategy for the industrial production of D-psicose (Figure 1).

Figure 1: The engineering schematic diagram of the project design
Figure 1. The engineering schematic diagram of the project design

Construction Design

Selection of novel NtDAEs was conducted in the NCBI database. The genes encoding DAE fused with 6×His-tag at its C-terminus were optimized and synthesized. The plasmid map was constructed by SnapGene software (Figure 2). NtDAE was inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI sites, and the recombinant plasmids pPICZαA-NtDAE were further transformed into E. coli DH5α.

Figure 2: The plasmid map of pPICZαA-NtDAE
Figure 2. The plasmid map of pPICZαA-NtDAE

Experimental Approach

We transferred the plasmid pPICZαA-NtDAE into E. coli DH5α and did single clone verification. Figure 3-A shows the PCR products were all highlighted between 750 and 1000 bp, which was consistent with the length of the target gene. Figure 3-B shows the E. coli plates we cultured and the location of samples for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 3-C showed the DNA sequence has no mutation. The recombinant construct was analyzed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-NtDAE.

Figure 3: Single clone verification and sequencing comparison of pPICZαA-NtDAE
Figure 3. The single clone verification and sequencing comparison of plasmid pPICZαA-NtDAE (E. coli DH5α). Note: NtDAE: 855 bp

Purification and SDS-PAGE

1. The transformed plate colony diagram (Pichia pastoris GS115)

The recombinant plasmids were transformed into Pichia pastoris GS115 for expression. Each kind of plasmid was cultured in three separate petri dishes. All three plates successfully developed colonies in Figure 4, which verified the plasmid (pPICZαA-NtDAE) had successfully transformed into Pichia pastoris GS115.

Figure 4: The transformed plate colony diagram of pPICZαA-NtDAE
Figure 4. The transformed plate colony diagram of pPICZαA-NtDAE (Pichia pastoris GS115)

2. The colony PCR identification (Pichia pastoris GS115)

A single colony containing the recombinant plasmid was cultured in medium containing antibiotics (100 mg/mL Zeocin) at 30°C. Next, the transformation status was verified 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 Figure 5. The result indicates successful transformation of Pichia pastoris GS115.

Figure 5: The colony PCR identification of pPICZαA-NtDAE
Figure 5. The colony PCR identification of pPICZαA-NtDAE (Pichia pastoris GS115)

3. SDA-PAGE detection

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 NtDAE was purified by Ni2+ affinity chromatography. The protein was detected by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). In both Figure 6 A and B, there’s protein falling between 34 kDa to 43 kDa. The coherence between target protein size and the observed bands indicates successful protein expression at 24 hours.

Figure 6: The SDA-PAGE detection of the products in the 24h
Figure 6. The SDA-PAGE detection of the products in the 24h. A. SDA-PAGE detection of crude protein extract. B. Purification of the recombinant His6-tagged protein. Note: NtDAE: 37.9 kDa

Next, the same steps were taken to process the 96-hour protein sample. In Figure 7-A, the purified protein fell between 34 kDa to 43 kDa. The coherence between target protein size and the observed bands indicates successful protein expression at 96 hours. The expression level of proteins can be characterized by intden values. The bands of the target protein were cut and used with ImageJ software to calculate the intden values of the protein bands (Figure 7-B). 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 the intden value of protein. In Figure 7-C, the intden value of NtDAE was significantly higher than that of CK (GS115), TcDAE, TtDAE, and DsDAE. It indicates that the protein expression level of NtDAE is higher than that of other groups. The protein expression of TcDAE and TtDAE was similar. The intden value of DsDAE compared with TcDAE and TtDAE, and the protein expression level of DsDAE is the lowest. However, the intden value of DsDAE, TcDAE, TtDAE, and NtDAE was significantly higher than those in the control group (GS115), showing that the protein expression of DsDAE, TcDAE, TtDAE, and NtDAE is successful.

Figure 7: The protein expression of TcDAE, TtSAE, NtDAE, and DsDAE at 96 hours
Figure 7. 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 ImageJ. C represents the intden value of different proteins, GS115 represents Pichia pastoris GS115.

Characterization/Measurement

1. Production of D-psicose

Using 10 mg/mL D-fructose as the substrate, we added 0.3 μmol of purified recombinase NtDAE and CK-GS115, 1 mmol/L CoCl2. Then each recombinant DAE was reacted at 40, 50, 60, 70, and 80 degrees for 10 minutes, and boiled for 10 minutes. 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 with a confidence interval of 0.05%. HPLC detection conditions: fixed phase is 2695 HPLC Waters Sugar-Pak I sugar column, and the mobile phase is ultrapure water. The flow rate is 0.4 mL/min, and the 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 NtDAE proteins can catalyze D-fructose to generate D-psicose.

Table 1. Production of D-psicose at different temperatures
Temperature (°C) 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
NtDAE 2.557 2.656 2.860 3.470 2.608

In Figure 8, the production of D-psicose in the NtDAE groups showed a trend of first increasing and then decreasing. The NtDAE groups at 70 degrees had the highest D-psicose production, indicating that NtDAE proteins are active and have the highest activity at 70 degrees.

Figure 8: Line graph of D-psicose production at different temperatures
Figure 8. Line graph of D-psicose production at different temperatures.

Comparing the D-psicose production of TcDAE, TtDAE, NtDAE, and DsDAE groups, it was found that the D-psicose productivity of the NtDAE group was significantly higher than that of the TcDAE, TtDAE, and DsDAE groups (Figure 9). There was no significant difference in the maximum yield among the TcDAE, TtDAE, and DsDAE groups. Therefore, the NtDAE group has the highest yield and the highest thermal stability.

Figure 9: Production of D-psicose at different temperatures
Figure 9. Production of D-psicose at different temperatures.

2. Conversion rate of D-psicose

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 was 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-allulose in TcDAE, TtDAE, NtDAE, and DsDAE groups
Group D-Fructose (mg) D-psicose (mg) Maximum Conversion Rate (%)
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.PTVA Method for Screening High Copy NtDAE and D-psicose Production

Inoculate the expression strain of D-allulose-3-isomerase from Novibacillus thermophilus into 3 mL YPD medium with an initial concentration of Zeocin 500 μg/mL. Next, 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 4 mg/mL Zeocin YPD medium, and the yield of D-allulose was measured using the same method by HPLC.

In Figure 10-A, the growth of the 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 NtDA E. In Figure 10-B, the D-psicose production of the NtDAE group is lower than that of the NtDAE-op group. The table in Figure 10-B shows that the conversion rate of the NtDAE group is 34.7%, and the conversion rate of the NtDAE-op group is 37.0%.

Figure 10: PTVA method for screening high copy NtDAE and D-psicose production
Figure 10. PTVA method for screening high copy NtDAE and D-psicose production.

Summary

Based on the above results, we conclude that DAE from Novibacillus thermophilus has the highest conversion rate of D-fructose to D-psicose, reaching 37%. It has been reported that the conversion rate of expression in E. coli was 29.7% [1]. Compared with the current research literature, we achieved a 6% increase.

At present, there are still some aspects of our study that need further exploration. Due to the particularity of Pichia pastoris, we could only select vectors like pPICZαA. The plasmid promoter is the alcohol oxidase promoter AOX1, which requires methanol induction. For safety reasons, we plan to replace the promoter in future studies, for example, with the PGK1 promoter or GAP promoter [2-3]. There have been related studies showing that fusion promoters can further optimize vector performance [3].

In this project, we selected four sources of DAE: Thermoclostridium caenicola (TcDAE), Novibacillus thermophilus (NtDAE), Thermogutta terrifontis (TtDAE), and Dorea sp. CAG317 (DsDAE). In the future, we aim to explore more DAE sources, such as D-psicose-3-epimerase (SfDAE) from Sinorhizobium fedii [4], and D-tagatose 3-epimerase from Thermotoga maritima [5]. Additionally, site-directed mutagenesis can be applied to optimize DAE from different sources to increase D-psicose yield, giving our products a competitive edge.

References

  1. Dong-Xu J, Chen-Yi S, Yi-Ting J, et al. Properties of D-allulose 3-epimerase mined from Novibacillus thermophilus and its application to the synthesis of D-allulose. Enzyme and Microbial Technology, 2021, 148109816-109816.
  2. Zhang Huijie, Liao Simin, Ling Xiaocui, etc. P. pastoris truncated PGK1 promoter combined with different terminators to regulate exogenous gene expression. Microbiology, 2022, 62 (07): 2642-2657. DOI: 10.13343/j.cnki.wsxb.20210653.
  3. Qian Kai, Zhang Jingjing, Wu Suping, etc. Expression and purification of GLP-1 analogues in Pichia pastoris using the GAP promoter. Chinese Journal of Bioengineering, 2015, 35 (05): 66-73. DOI: 10.13523/j.cb.20150510.
  4. Wu Mian. Study on the efficient secretory expression of D-psicose 3-epimerase by Bacillus subtilis to produce D-psicose. Tianjin University of Science and Technology, 2022. DOI: 10.27359/d.cnki.gtqgu.2022.000764.
  5. Qi Z, Zhu Z, Wang J-W, et al. Biochemical analysis and preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides. Microbial Cell Factories, 2017, 16.