RESULTS
The use of saccharides in the production and processing of food has proliferated as people’s demand for food flavor has increased. Excessive intake of saccharides may lead to increase in blood glucose level, which may threaten health; sugar substitutes such as aspartame may lead to intestinal flora disorders. As a new type of sugar substitute product, Thaumatin has the advantages of high sweetness and good stability. However, the traditional Thaumatin is mainly extracted from plants, which faces the problems of low yield and high production cost. This project proposes to improve the production efficiency of Thaumatin by inserting the Thaumatin protein gene into E. coli for bioculture.
Figure 1 Overview of Experimental Steps
We first configured LB liquid and solid medium. Then a large number of genes of PDI and Thaumatin-A/B/C/D were obtained by PCR amplification respectively. We obtained the fragments of the target genes by performing agarose gel electrophoresis. Figure 2 shows the agarose gel electrophoresis results of PCR for five clones. We can find out that the DNA fragment of PDI is approximately 1586 bp in length, Thaumatin-A is 1011 bp, Thaumatin-B is 759 bp, Thaumatin-C is 1062 bp and Thaumatin-D is 1125 bp, which is consistent with the predicted size. This indicates that the target gene was successfully amplified.
Figure 2 Agarose gel electrophoresis results of PCR for five clones
We used two different restriction endonucleases BamhI and HindIII to cleave the two cleavage sites on the plasmid, avoiding plasmid closure before insertion of the gene, and facilitating the insertion of the gene at a later stage. Then we performed DNA electrophoresis to make gel plates, and then DNA recovery to obtain the pRSFDuet-Linear Vector. We used homologous recombination technology to insert two different homologous recombinases (F1 and R1) at each end of the PDI gene and the plasmid, and then inserted the target gene into the plasmid to allow the homologous recombinases to bind to each other. Finally we got the plasmid with four Thaumatin genes and PDI respectively.
After homologous recombination, the recombinant plasmids were transformed into chemically competent E. coli DH5α cells via the heat shock method. Following overnight incubation on LB agar plates containing the appropriate antibiotic, several well-isolated colonies were observed (Figure 3), indicating successful transformation.
Figure 3 Agar plate showing DH5α colonies after transformation
To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in Figure 4, each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene.
Figure 4 Agarose gel electrophoresis results of Colony PCR identification for five clones
We extracted the plasmid and sent it to a biotechnology company for sequencing to verify the accuracy of the target gene sequences. As shown in Figure 5, the sequencing results are completely consistent with the target sequences, confirming the successful construction of our plasmid.
Figure 5 DNA sequencing results of recombinant plasmids
After LB media preparation and PCR-based amplification/recovery, E. coli Origami2(DE3) was transformed with five recombinant plasmids. After incubation, as shown in Figure 6, numerous single colonies of E. coli Origami2(DE3) grew on the medium. These well-separated, round, and milky-white colonies indicated successful transformation and stable propagation of the engineered bacteria. This laid a foundation for subsequent large-scale liquid culture expansion and induced osmotic expression of the target protein, ensuring sufficient bacterial biomass for subsequent protein expression experiments.
Figure 6 Agar plate showing BL21 colonies after transformation
After ultrasonic lysis of E. coli Origami2(DE3) expressing Thaumatin-A/B/C/D, the samples were separated into supernatant and precipitate fractions, which were then analyzed by SDS-PAGE (Figure 7). In the supernatant lanes (left of Marker M), distinct protein bands corresponding to the expected molecular weight (~38 kDa) were observed for Thaumatin-A/B/C/D, indicating that the target proteins were successfully extracted into the soluble fraction via ultrasonic lysis. The precipitate lanes (right of Marker M) showed relatively weaker and less prominent bands at the ~38 kDa position, suggesting that most of the target proteins remained in the soluble supernatant after lysis. The clear ~38 kDa bands in the supernatant confirmed that ultrasonic lysis effectively released the target proteins, providing soluble crude protein samples for subsequent purification and analysis.
Figure 7 SDS-PAGE analysis of crude protein
After ultrasonic lysis and centrifugation, the supernatant containing crude proteins was subjected to Ni-NTA affinity purification. The resulting elution fractions were analyzed by SDS-PAGE. The results are shown in Figure 8. In the supernatant lanes (left of Marker M), complex protein bands were observed, indicating a mixture of target proteins and other impurities prior to purification. In the elution lanes (right of Marker M), distinct bands at ~38 kDa, corresponding to Thaumatin-A/B/C/D, were detected.
This result confirmed that Ni-NTA affinity purification effectively enriched the target proteins. The clear, concentrated ~38 kDa bands in the elution fractions demonstrated successful separation of the target proteins from most impurities, providing purified protein samples for subsequent experiments.
Figure 8 SDS-PAGE analysis After Protein Elution
TLC was employed to analyze the monosaccharide expression resulting from the action of Thaumatin-A and Thaumatin-B. As shown in Figure 9, 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 9 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 10, 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.
β-1,3-glucanase hydrolyzes laminarin into reducing sugars. These sugars react with DNS reagent (3,5-dinitrosalicylic acid) in boiling water, forming a colored complex. For the samples (reaction systems containing Thuam proteins), their absorbance values were measured and substituted into the standard curve equation to calculate glucose concentrations. This allowed for the quantification of glucose production, facilitating the analysis of the ability of Thuam proteins to catalyze substrate degradation and glucose production, thus completing the functional verification from the perspective of glucose content.
Figure 10 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 11 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 12 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 13 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 14, 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 14 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 15A, with each sample group forming distinct clusters, indicating significant differences in taste profiles between samples. Sweetness values for different samples were shown in Figure 15B. 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 15 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. The model of the sweet taste receptor protein was downloaded from the Protein Data Bank (PDB). The model of the sweet protein TLP was predicted using AlphaFold2. And the molecular docking between the sweet protein TLP and the sweet taste receptor protein was performed using the Z-DOCK online tool.
For TLP-A, the predicted structure contained 12 β-sheets and 6 α-helices, presenting an overall compact globular conformation. The conformation of surface flexible loop regions was reasonable, and the predicted local distance difference test (pLDDT) scores were all > 85, indicating high structural reliability. For TLP-B, the predicted structure consisted of 12 β-sheets and 2 α-helices, also showing an overall compact globular conformation. The surface flexible loop regions had a reasonable conformation, and the pLDDT scores were equally > 85, confirming the reliability of its structural prediction.
As shown in Figure 16, 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 16 The interaction between the sweet taste receptor and TLP-A
As shown in Figure 17, 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 17 The interaction between the sweet taste receptor and TLP-B
To optimize taste, we will improve potential aftertaste issues through protein structure modification or compounding techniques. In terms of safety control, we will conduct specific allergen testing to identify the sources of potential allergens.
We will test solubility and thermal stability, specifically measure solubility and dissolution rate under different solvents, temperatures, and pH conditions. Meanwhile, we will also test taste stability in different food matrices.
