ENGINEERING
Currently, many people suffer from health issues like obesity and diabetes due to excessive sucrose intake. To reduce sugar consumption, many turn to sugar substitutes such as aspartame or erythritol; however, consuming these traditional sugar substitutes often leads to side effects like dizziness and nausea.
In nature, there exists a protein that produces sweetness by binding to human receptors. We aimed to express the gene encoding this protein in a bacterial plasmid. We used bacterial homologous genes that are highly similar to the plant gene—specifically, four genes named Thaumatin A/B/C/D. Additionally, we included a fragment of the PDI (Protein Disulfide Isomerase) gene to promote proper protein folding by facilitating the correct formation of disulfide bonds, thereby enhancing proper protein expression.
Design
We selected the pRSFDuet plasmid with dual promoters to achieve the simultaneous expression of two proteins (Thaumatin homologous protein and PDI protein). The pRSFDuet plasmid vector contains two independent multiple cloning sites, each equipped with a T7 promoter, a ribosomal binding site, and a transcription terminator, enabling efficient regulation of the synchronous expression of two target genes in the same host cell. Additionally, it has a kanamycin resistance marker, which facilitates the screening and identification of positive clones.
Figure 1 The map of pRSFDuet plasmid
We successfully inserted the PDI gene fragment into the pRSFDuet plasmid via double digestion and homologous recombination techniques. The PDI protein catalyzes the formation of protein disulphide bonds, which results in the correct folding of the protein, allowing the Thaumatin A/B/C/D proteins to successfully bind to the human sweetness receptor and generate a sweet taste.
Figure 2 The map of pRSFDuet-PDI
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The PDI gene fragment (1568 bp) was amplified using PCR. Figure 3 showed the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of PDI is approximately 1568 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 3 Gel electrophoresis of PCR for PDI gene
Both the gene and the pRSFDuet vector were digested with BamHI and HindIII restriction enzymes to generate compatible sticky ends, and then assembled using the homologous recombination enzyme. The ligation product was transformed into E. coli DH5α competent cells via heat shock to obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing.
Figure 4 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI
Design
We selected the pRSFDuet-PDI recombinant plasmid as the base vector, and inserted the Thaumatin-A gene fragment via double digestion and homologous recombination techniques to construct a new recombinant plasmid.
Figure 5 The map of pRSFDuet-PDI-Thaumatin-A
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The Thaumatin-A gene (1011 bp) was amplified by PCR. Figure 6 showed the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of Thaumatin-A gene is approximately 1011 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 6 Gel electrophoresis of PCR for Thaumatin-A
The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-A by homologous recombination. The ligation product was transformed into E. coli DH5α competent cells. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing.
Figure 7 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-A
Design
The design is pragmatically like that of Part 2, except we used Thaumatin-B, in place of Thaumatin-A.
Figure 8 The map of pRSFDuet-PDI-Thaumatin-B
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The Thaumatin-B gene (759 bp) was amplified by PCR. Agarose gel electrophoresis was also used to determine whether the PCR-amplified gene fragment was Thaumatin-B. As Figure 9 showed, the DNA fragment of Thaumatin-B gene is approximately 759 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 9 Gel electrophoresis of PCR for Thaumatin-B
The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-B by homologous recombination. The ligation product was transformed into E. coli DH5α competent cells via heat shock to obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing. As shown in Figure 10, the Thaumatin-B gene fragment was successfully ligated to the pRSFDuet-PDI vector without any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-B plasmid.
Figure 10 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-B
Design
The design is pragmatically like that of Part 2, except we used Thaumatin-C, in place of Thaumatin-A.
Figure 11 The map of pRSFDuet-PDI-Thaumatin-C
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The Thaumatin-C gene (1062 bp) was amplified by PCR. Figure 12 showed the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of Thaumatin-C gene is approximately 1062 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 12 Gel electrophoresis of PCR for Thaumatin-C
The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-C by homologous recombination. The ligation product was transformed into E. coli DH5α competent cells via heat shock to obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing. As shown in Figure 13, the Thaumatin-C gene fragment was successfully ligated to the pRSFDuet-PDI vector without any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-C plasmid.
Figure 13 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-C
Design
The design is pragmatically like that of Part 2, except we used Thaumatin-D, in place of Thaumatin-A.
Figure 14 The map of pRSFDuet-PDI-Thaumatin-D
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The Thaumatin-D gene (1125 bp) was amplified by PCR. Figure 15 showed the agarose gel electrophoresis results of PCR. We can find out that the DNA fragment of Thaumatin-D gene is approximately 1125 bp in length, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 15 Gel electrophoresis of PCR for Thaumatin-D
The pRSFDuet-PDI plasmid was linearized and assembled with the gene fragment of Thaumatin-D by homologous recombination. The ligation product was transformed into E. coli DH5α competent cells via heat shock to obtain the recombinant plasmid. Colonies were screened using colony PCR, and the band sizes were confirmed through gel electrophoresis and DNA sequencing. As shown in Figure 16, the Thaumatin-D gene fragment was successfully ligated to the pRSFDuet-PDI vector without any apparent mutations, confirming the successful construction of the pRSFDuet-PDI-Thaumatin-D plasmid.
Figure 16 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-D
The confirmed plasmids were transformed into E.coli Origami2(DE3) for protein expression. IPTG was added to induce expression, and the culture was harvested after 20 hours. Cell lysis was performed using a sonicator under low-temperature conditions to prevent protease denaturation. Purification was carried out using nickel affinity chromatography. The column was washed with buffer A (low imidazole) and eluted with buffer B (high imidazole). Collected fractions including lysate, flow-through, and elution were analyzed via SDS-PAGE.
As shown in Figure 17, distinct protein bands were observed at approximately 38 kDa for all target proteins. This result is consistent with the expected molecular weight, indicating that these Thaumatin proteins were successfully expressed and purified in the elution fractions under the current experimental conditions.
TLC was employed to analyze the monosaccharide expression resulting from the action of Thaumatin-A and Thaumatin-B. As shown in Figure 18, distinct bands were observed in the lanes, within the region marked by the red box. This TLC-based detection demonstrated that Thaumatin-A and Thaumatin-B can mediate the breakdown of polysaccharide substrates to produce monosaccharide-related products, successfully achieving the functional verification of monosaccharide expression at the enzymatic activity level.
Figure 18 TLC Detection of Monosaccharide Expression
To quantify glucose levels, the DNS method was used. A standard curve was constructed by measuring the absorbance (at 540 nm) of DNS-reacted glucose solutions with known concentrations. As shown in Figure 19, the linear equation is y = 0.9596x-0.0189 with a high correlation coefficient R2 = 0.9907, indicating a good linear relationship between absorbance and glucose concentration in the tested range.
Figure 19 Glucose Standard Curve
The Single-enzyme activity results showed that Thaumatin-A had the highest enzyme activity, followed by Thaumatin-B, Thaumatin-D, and Thaumatin-C.
Figure 20 Graphs of single-enzyme activity comparison
In the dual-enzyme combinations, the mixture of Thaumatin-C&D displayed the highest activity, followed by Thaumatin-A&C, Thaumatin-A&B, Thaumatin-A&D, Thaumatin-B&D, and Thaumatin-B&C.
Figure 21 Graphs of dual-enzyme activity comparison
In the triple-enzyme combinations, the mixture of Thaumatin-A&B&D displayed the highest activity, followed by Thaumatin-A&B&C, and Thaumatin-B&C&D.
Figure 22 Graphs of triple-enzyme activity comparison
Finally, the above three combinations with the best activity were selected and compared with the mixed system containing all four enzymes. As shown in Figure 23, the mixture of Thaumatin-A&B&C&D displayed the highest activity, followed by Thaumatin-C&D, Thaumatin-A&B&D, and Thaumatin-A.
Figure 23 Graphs of optimal combinations comparison
To characterize the taste properties of samples, electronic tongue detection was performed.The differences in taste characteristics among different samples were shown in Figure 24A, with each sample group forming distinct clusters, indicating significant differences in taste profiles between samples. Sweetness values for different samples were shown in Figure 24B. The 4% Sucrose group served as a control with extremely low sweetness. Thaumatin-A had the highest sweetness, followed by Thaumatin-C, Thaumatin-D, and Thaumatin-B. These results indicate that Thaumatin proteins can significantly enhance the sweetness of the system, with varying degrees of sweetness-enhancing effects among different Thaumatin variants.
Figure 24 Result of electronic tongue detection
To investigate the binding mode between sweet proteins and sweet taste receptor proteins, we performed AlphaFold modeling and protein-protein molecular docking. As shown in Figure 25, the sweet protein TLP-A formed 5 sets of hydrogen bonds with TAS1R2. The amino acid residues in TLP-A form hydrogen bonded with S87, D406, and S408 of TAS1R2, respectively.
Figure 25 The Interaction between the Sweet Taste Receptor and TLP-A
As shown in Figure 26, the sweet protein TLP-B forms 3 sets of hydrogen bonds with TAS1R2. The amino acid residues in TLP-B form hydrogen bonded with W341, N368, and D364 of TAS1R2, respectively. The TAS1R2 region involved in the interaction is the key binding site for the combination of sweet ligand proteins and sweet molecules.
Figure 26 The Interaction between the Sweet Taste Receptor and TLP-B
1. Different PCR and DNA assembly conditions can affect fragment amplification and DNA assembly. We conducted multiple troubleshooting steps by adjusting the amounts of primers and templates, as well as the PCR reaction program. All the target genes were successfully amplified, and the successful construction of the recombinant plasmids were confirmed by sequencing and electrophoresis analyses.
2. Clear bands were observed at ~38 kDa in the supernatant lanes of Thaumatin-C/D, indicating that the target protein is expressed in a soluble form. For Thaumatin-A/B, it is recommended to improve their solubility by adjusting the expression conditions: methods such as lowering the temperature (e.g., 30°C/25°C) with a slight extension of induction time, or optimizing the inducer (testing different IPTG concentrations or switching to lactose) can be adopted. Ultimately, verification by SDS-PAGE should be performed to obtain stronger ~38 kDa bands in their supernatants.
3. Among the enzyme activity assays, the Thaumatin-A&B&C&D combination exhibited the highest activity. This demonstrates the synergistic effect formed through functional complementation between different Thaumatin proteins, which can enhance the catalytic capacity for substrates, highlighting the significant advantages of multi-component combined use in improving enzymatic reaction efficiency.
