Part:BBa_K5528006

pPICZαA-TtDAE

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]

Engineering Principle

D-fructose can be converted into D-psicose by D-psicose 3-epimerase 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

Construction Design

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

Figure 2. The plasmid map of pPICZαA-TtDAE

Experimental Approach

We transferred the plasmid pPICZαA-TtDAE 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 analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named pPICZαA-TtDAE (Figure 3).

Figure 3. Single clone verification and sequencing comparison of plasmid pPICZαA-TtDAE (E. coli DH5α). TtDAE: 873 bp

Purification and SDS-PAGE

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

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

Figure 4. The transformed plate colony diagram of pPICZαA-TtDAE (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, 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 Figure 5. The result indicates successful transformation of Pichia pastoris GS115.

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

3. SDA-PAGE detection of the products

After induction, we induced sampling at 24 hours. The bacterial cells were lysed by sonication in phosphate buffer. The recombinant His6-fused TtDAE was purified by Ni²⁺ affinity chromatography. The protein was detected by 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). In Figure 6, the protein fell 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. A. SDA-PAGE detection of crude protein extract. B. Purification of the recombinant His6-tagged protein. Note: TtDAE: 37.3 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 TtDAE and CK (GS115), and the ordinate was the intden value of protein. In Figure 7-C, the intden value of TtDAE was significantly higher than that of CK (Pichia pastoris GS115), showing that the protein expression of TtDAE was successful.

Figure 7. The protein expression of TtDAE 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 TtDAE 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 by Weipu Biological Company, and the confidence interval was 0.05%. HPLC detection conditions: the 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 TtDAE 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
TtDAE	1.174	1.666	2.317	2.017	1.585

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 8. Moreover, the TtDAE groups at 60 degrees showed the highest production of D-psicose, indicating that the enzyme activity of TtDAE is highest at 60 degrees.

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

2. Conversion rate of D-psicose

Using D-fructose as a substrate, TtDAE 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 the TtDAE group are 23.2%.

Table 2. Maximum conversion rate of D-allulose in TtDAE group

Group	D-Fructose (mg)	D-psicose (mg)	Maximum Conversion Rate (%)
Blank control	10	0	0%
Pichia pastoris GS115	10	0	0%
TtDAE	10	2.317	23.2%