Results
1. Construction Results
1.1 Construction of DTE Enzyme Expression Systems
We constructed different DTE enzyme expression systems following this logic:
We first obtained the target fragment via PCR, added BsaI restriction sites at both ends, then constructed the recombinant plasmid using Golden Gate technology. Following antibiotic screening, potential colonies were selected via colony PCR, and sequencing confirmed correct plasmid ligation and transformation outcomes.
Colony clones expressing the three DTE enzyme systems were successfully obtained.
Colonies with appropriate fragment sizes were submitted for sequencing. The results are shown below, indicating normal sequences suitable for subsequent experiments.
1.2 Construction of an RNA-pepper-based DTE enzyme activity characterization system
Using electroporation, the DTE enzyme expression system and the RNA-pepper-based allulose biosensor were introduced into E. coli DH5α to construct an RNA-pepper-based DTE enzyme activity characterization system. The specific procedure is as follows:
Take overnight-cultured E. coli and transfer it into fresh medium for 4 hours of cultivation;
Collect the transferred E. coli, wash three times with 10% glycerol, and aliquot 100 μL into sterile centrifuge tubes to prepare E. coli competent cells.
In a laminar flow hood, add all constructed plasmids to the thawed competent E. coli. Gently mix, then transfer to a pre-chilled electroporation cup placed on ice. Wipe moisture from both sides of the cup before placing it in the electroporator for transformation.
Add 1 mL LB medium to the electroporation cup, vortex thoroughly, and transfer to centrifuge tubes. Incubate at 37°C, 200 rpm for 1 hour to allow recovery.
Centrifuge the recovered culture at 25°C and 4000 rpm for 2 minutes. Discard most of the supernatant, resuspend the remaining cells, and plate them onto LB solid agar plates containing the corresponding antibiotic. Invert the plates and incubate at 37°C for 16 hours.
Select moderately sized colonies growing on the corresponding antibiotic plates for colony PCR verification. Results are as follows:
Some colonies yielded correct PCR verification results and were suitable for further experiments. With this, the construction of all three systems was completed, enabling progression to the subsequent efficacy characterization phase.
2. Test Results
2.1 DTE enzyme expression system detection and DTE enzyme catalytic activity experimental results
We first measured the growth curves of strains expressing different DTE enzymes. We observed that the growth rates of different strains were largely similar, as the sizes of these three DTE enzymes were nearly identical.
To validate the system's ability to express DTE enzymes, we detected the expression levels of the three DTE enzymes via RT-qPCR. E. coli DH5α without the expression system served as the blank control. The detection results for all three expression systems were significantly higher than the control group.
Next, we precisely quantified allulose content in the catalytic system using high-performance liquid chromatography (HPLC). The HPLC instrument configuration was as follows:
Column: Installed Waters Sugar-PAK I sugar-specific separation column.
Mobile phase: Wahaha purified water containing 50 mg/L ethylenediaminetetraacetic acid (EDTA), flow rate set at 0.4 mL/min.
Column temperature: Set to 80°C.
Detector: A refractive index detector was selected.
Injection volume: 5 μL per injection.
We prepared 50 mg/mL monosaccharide solutions of D-fructose and D-allulose to determine the retention times of their chromatographic peaks. As shown in the schematic diagram below, the retention time for D-fructose is 14.5 min, while that for D-allulose is approximately 15 min:
Next, to quantitatively calculate the catalytic efficiency of DTEK, we prepared mixed sugar solutions containing D-fructose and D-allulose at concentrations of 0.0313, 0.125, 0.25, 0.5, 0.75, and 1.5 g/L of D-fructose and D-allulose mixed sugar solutions. Standard curves for D-fructose and D-allulose were plotted with concentration on the x-axis and peak area on the y-axis.
The standard curves exhibit good fit.
Finally, wild-type E. coli DH5α was selected as the control group, and recombinant E. coli DH5α (pSEVA341-DTE-WT) as the experimental group for separate cultivation.
After adjusting the bacterial density to be consistent, the cultures were incubated at 37°C for 2 hours. During this period, 300 mM fructose solution was added to the recombinant E. coli DH5α (pSEVA341-DTE-WT) culture medium for induction. After induction, supernatants were collected from both control and experimental groups. Each supernatant underwent 50-fold dilution to ensure the diluted fructose and allulose concentrations fell within the standard curve range (0.0313–1.5 g/L). Diluted samples were filtered through a 0.22 μm aqueous phase membrane before injection for analysis. Chromatograms were recorded. Calculate the concentrations of D-fructose and D-allulose in the samples based on the standard curve.
&emspThe following formula was used to calculate D-allulose conversion rate, total D-fructose conversion rate, consumption rate, and other data reflecting the catalytic efficiency of DTE enzyme..
D-Allulose Conversion Rate (F) Calculation Formula
\[
F = \frac{c_a}{c_{f_0}} \times 100\%
\]
Where: D = D-allulose conversion rate; ca = post-reaction D-allulose concentration (g/L); cf0 = initial D-fructose concentration (g/L).
D-Fructose Total Conversion Rate (D) Calculation Formula
\[
D = \frac{c_{f_0} - c_f}{c_{f_0}} \times 100\%
\]
Where: D represents the total D-fructose conversion rate; cf₀ denotes the initial D-fructose concentration (g/L); cf indicates the D-fructose concentration in the supernatant after the whole-cell reaction (g/L).
Formula for D-fructose consumption rate (G)
\[
G = D - F
\]
The fructose consumption rate G represents the difference between the total D-fructose conversion rate and the D-allulose conversion rate, indicating the proportion of D-fructose diverted into alternative metabolic pathways.
However, liquid chromatography analysis revealed that both the control blank E. coli and E. coli expressing the DTE enzyme system exhibited indistinct allose peaks. Analysis indicated that insufficient bacterial biomass led to low allose production levels below the detection limit of liquid chromatography.
2.2 Experimental results of the RNA-pepper-based allulose biosensor
Following construction and transformation of the pet28-porin-PsiRA-pepper plasmid, induction was performed at various allulose concentrations. The fluorescence intensity of the pepper-HBC dye complex was measured after 2 hours, yielding the following results:
It can be observed that as the concentration of the allulose inducer increases, the fluorescence intensity of the pepper-HBC dye complex also gradually increases. Furthermore, the fluorescence intensity exhibits an approximate S-shaped curve relationship with the inducer concentration, consistent with the expected results.
2.3 Experimental results of the RNA-pepper-based DTE enzyme activity characterization system
By transferring Pet28-porin-psiRA-pepper and pSEVA341-porin-DTE into DH5α and validating them, we successfully constructed the genetic circuit diagram shown below: As DTE enzyme catalyzes fructose conversion to allulose, exposure to PsiR inhibits pPsiA expression, leading to pepper expression. The fluorescence intensity of the pepper-HBC complex indirectly reflects DTE enzyme catalytic activity.
First, we explored the optimal operating conditions required for this activity characterization system. To determine the optimal fructose induction concentration, we incubated the cellular catalytic system at 100, 200, 300, 400, and 500 mM/L for 4 hours and measured fluorescence intensity. We observed that beyond 300 mM/L, further increases in fructose concentration did not significantly alter fluorescence intensity. Therefore, subsequent experiments employed 300 mM/L fructose for catalysis.
Similarly, to determine the optimal reaction duration, we reacted the Pet28-porin-psiRA-pepper and pSEVA341-porin-DTE-WT systems at 300 mM/L for 2, 4, and 6 hours, obtaining fluorescence intensity data. Considering the degradation curve of pepper mRNA obtained in the dry experiment section, we concluded that fluorescence intensity beyond 6 hours of reaction no longer accurately reflects the actual allulose concentration. Therefore, in subsequent measurements, we will continue to monitor fluorescence intensity changes during the 6-hour catalytic process.
To characterize the catalytic performance of different DTE enzymes, we measured the fluorescence intensity of strains expressing various DTE enzymes at 2h, 4h, and 6h under 300 mM/L fructose conditions, as shown below:
It can be observed that both the newly designed DTE1 and DTE2 exhibit catalytic activity. After 2 hours of catalysis, the activity differences among the various DTE enzymes are not significant. However, at 4 hours, the fluorescence intensity in both the DTE1 and DTE2 groups decreases to some extent. This may be because pepper itself is unstable, and the catalytic rates of DTE1 and DTE2 are not high. Additionally, converting fructose to allulose via DTE enzymes requires time before allulose interacts with the pepper expression inhibitor, leading to reduced pepper expression during this period. At 6 hours, fluorescence intensity increased across all groups. Since the DTE enzyme's conversion of fructose to allulose is reversible, it can be inferred that DTE1 likely exhibited higher conversion efficiency at this time point, while no significant activity difference was observed between DTE2 and the wild-type (WT).