Base Pairs: 1568 bp

Origin: Often derived from microbial or eukaryotic sources for heterologous expression, playing a role in assisting protein folding within expression systems

Description: PDI（Protein Disulfide Isomerase）

Properties: A crucial molecular chaperone, catalyzes the formation and isomerization of disulfide bonds in proteins, helps misfolded proteins refold correctly, and enhances the solubility and functional activity of target proteins (such as Thaumatin-A). It has a characteristic domain structure related to its chaperone and catalytic functions.

Figure 1 Gene map of PDI

Usage and Biology: PDI can interact with newly synthesized or misfolded proteins, recognizing incorrect disulfide bond configurations. Through its disulfide bond isomerase activity, it breaks and reforms disulfide bonds, guiding proteins to fold into their correct three-dimensional structures. In the prokaryotic expression system of recombinant proteins (such as Thaumatin-A expressed in E. coli), the co-expression of PDI can effectively improve the solubility of target proteins, reduce the formation of inclusion bodies, and ensure that the target proteins have normal biological functions. It is widely used in the fields of protein engineering, biotechnology, and pharmaceutical research to optimize the expression and production of functional proteins.

Cultivation: A single colony containing the PDI plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the PDI coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 2 Result of gel electrophoresis of PDI

Base Pairs: 1011 bp

Origin: Microbial-derived thaumatin-like protein

Description: Thaumatin-A

Properties: A homologous protein in the thaumatin family, with a unique amino acid sequence compared to other members of the family. It can specifically bind to sweet taste receptors to exert sweetness, and its sweetness multiple relative to sucrose has characteristics that make it potentially applicable as a natural sweetener.

Figure 3 Gene map of Thaumatin-A

Usage and Biology: Thaumatin-A protein can specifically bind to sweet taste receptors in human taste buds. Through interactions between its specific amino acid residues and the receptor structure, it activates the receptors, ultimately triggering the release of the neurotransmitter ATP. This signal is transmitted via neural pathways to the brain’s taste cortex, enabling the perception of sweetness. As a biosynthetic alternative for developing natural sweeteners, it can be used to produce low-calorie, high-sweetness natural sweeteners, meeting the demand for healthy food ingredients. It can also be combined with other sweeteners (such as stevioside) to modulate flavor and improve taste experience.

Cultivation: A single colony containing the Thaumatin-A plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the Thaumatin-A coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 4 Result of gel electrophoresis of Thaumatin-A

Base Pairs: 759 bp

Origin: Microbial-derived thaumatin-like protein

Description: Thaumatin-B

Properties: A homologous protein in the thaumatin family, highly similar to natural somatostatin in amino acid sequence, but with possible differences at certain key sites, leading to changes in function or sweetness, with a sweetness approximately 1600-3000 times that of sucrose.

Figure 5 Gene map of Thaumatin-B

Usage and Biology: Thaumatin-B protein is a kind of protein which has high similarity to natural Thaumatin protein. It can specifically bind to sweet taste receptors in human taste buds, primarily through its positively charged amino acids (such as lysine and arginine) binding to the negatively charged regions of the receptors, activating them, and ultimately releasing the neurotransmitter AT, which is transmitted via neural pathways to the brain’s taste cortex, which is ultimately recognised as sweetness. As a biosynthetic natural alternative to artificial sweeteners, it is used to produce low-calorie, high-sweetness natural sweeteners to meet the demand for healthy foods. It can be used in combination with other sweeteners (such as stevioside) to enhance flavour and improve taste.

Cultivation: A single colony containing the Thaumatin-B plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the Thaumatin-B coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 6 Result of gel electrophoresis of Thaumatin-B

Base Pairs: 1062 bp

Origin: Microbial-derived thaumatin-like proteins

Description: Thaumatin-C

Properties: Thaumatin-C is a sweet-tasting protein with extremely high sweetness, being 1600 times that of sucrose. It produces a sweet taste by binding to human taste receptors.

Figure 7 Gene map of Thaumatin-C

Usage and Biology: Thaumatin-C a high-potency natural sweetener (2000× sucrose, low-calorie), utilizes plasmid-encoded PDI for disulfide bond-dependent folding; it binds the T1R2/T1R3 sweetness receptor via G-protein signaling and is expressed inOrigami2(DE3) using a plasmid with T7 promoter, 6×His/S-Tag, KanR, and RSF ori, enabling applications in low-sugar foods, taste research, or bitterness masking.

Cultivation: A single colony containing the Thaumatin-C plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the Thaumatin-C coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 8 Result of gel electrophoresis of Thaumatin-C

Base Pairs: 1125 bp

Origin: Microbial-derived thaumatin-like proteins

Description: Thaumatin-D

Properties: Thaumatin-D is a sweet-tasting protein with extremely high sweetness, being 6000 times that of sucrose. It produces a sweet taste by binding to human taste receptors.

Figure 9 Gene map of Thaumatin-D

Usage and Biology: Thaumatin-D is the target expression product. It possesses the characteristics of high sweetness and low calories, making it suitable for use as a natural sweetener in various industries, including the food industry. Thaumatin-D can bind to sweet taste receptors on the tongue and activate relevant taste signal transduction pathways. Its binding mode to sweet taste receptors is unique and different from that of traditional carbohydrate sweeteners. The specific structure of Thaumatin-D enables it to interact with certain subunits of sweet taste receptors, triggering conformational changes, and then activating the downstream G-protein-coupled receptor signal pathway. Eventually, the sweet taste signal is transmitted to the brain, resulting in the perception of sweetness.

Cultivation: A single colony containing the Thaumatin-D plasmid was inoculated into liquid LB medium supplemented with kanamycin and incubated overnight at 37 °C with constant shaking. Plasmid DNA was subsequently extracted and used as the template for PCR amplification of the Thaumatin-D coding sequence using gene-specific primers under optimized thermal cycling conditions. The PCR products were analyzed by agarose gel electrophoresis to confirm the presence and expected size of the amplified fragment.

Figure 10 Result of gel electrophoresis of Thaumatin-D

Composition: pRSFDuet backbone; PDI gene fragment.

Apparatus used: pRSFDuet plasmid, PDI gene fragment., restriction endonucleases, Homologous recombination enzyme.

Figure 11 Plasmid map of pRSFDuet-PDI

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression. 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.

The PDI gene fragment (1568 bp) was amplified using PCR. Figure 12 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 12 Result of gel electrophoresis of PDI

Both the gene and the pRSFDuet vector were digested with 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. To verify the presence of the target gene, five individual colonies were randomly selected for colony PCR. As shown in the gel electrophoresis results (Figure 13B), each colony produced a DNA band of the expected size, confirming successful plasmid construction and correct insertion of the target gene. And the band sizes were then confirmed through DNA sequencing. As shown in Figure 13C, the inserted gene fragments matched the designed sequences exactly, with no frameshift mutations or base substitutions, indicating successful and accurate cloning of the target genes.

Figure 13 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI

Composition: pRSFDuet backbone; PDI gene fragment; Thaumatin-A gene fragment

Apparatus used: pRSFDuet plasmid, PDI gene fragment, Thaumatin-A gene fragment, restriction endonucleases, Homologous recombination enzyme.

Figure 14 Plasmid map of pRSFDuet-PDI-Thaumatin-A

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression. 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.

The Thaumatin-A gene (1011 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-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 15 Result of gel electrophoresis of 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 16 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-A

Composition: pRSFDuet backbone; PDI gene fragment; Thaumatin-B gene fragment

Apparatus used: pRSFDuet plasmid, PDI gene fragment, Thaumatin-B gene fragment, restriction endonucleases, Homologous recombination enzyme.

Figure 17 Plasmid map of pRSFDuet-PDI-Thaumatin-B

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression. 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.

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 18 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 18 Result of gel electrophoresis of 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 19, 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 19 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-B

Composition: pRSFDuet backbone; PDI gene fragment; Thaumatin-C gene fragment

Apparatus used: pRSFDuet plasmid, PDI gene fragment, Thaumatin-C gene fragment, restriction endonucleases, Homologous recombination enzyme.

Figure 20 Plasmid map of pRSFDuet-PDI-Thaumatin-C

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression. 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.

The Thaumatin-C gene (1062 bp) was amplified by PCR. Figure 21 showed the agarose gel electrophoresis results of PCR. 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 21 Result of gel electrophoresis of 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 22, 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 22 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-C

Composition: pRSFDuet backbone; PDI gene fragment; Thaumatin-D gene fragment

Apparatus used: pRSFDuet plasmid, PDI gene fragment, Thaumatin-D gene fragment, restriction endonucleases, Homologous recombination enzyme.

Figure 23 Plasmid map of pRSFDuet-PDI-Thaumatin-D

Engineering Principle:

As a vector with dual promoters, the pRSFDuet plasmid enables the simultaneous expression of two proteins. It contains the RSF origin of replication (ori) to ensure stable replication and also includes promoter/regulatory elements such as lacIq and T7 for regulating gene expression. 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.

The Thaumatin-D gene (1125 bp) was amplified by PCR. Figure 24 showed the agarose gel electrophoresis results of PCR. 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 24 Result of gel electrophoresis of 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 25, 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 25 Colony growth, gel electrophoresis result and DNA sequencing of pRSFDuet-PDI-Thaumatin-D

Protein expression was induced with IPTG (1 mM) in E. coli Origami2(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant papain protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 26, 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.

Figure 26 SDS-PAGE results of all target proteins

TLC was employed to analyze the monosaccharide expression resulting from the action of Thaumatin-A and Thaumatin-B. As shown in Figure 27, 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 27 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 28, 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 28 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 29 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 30 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 31 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 32, 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 32 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 33A, with each sample group forming distinct clusters, indicating significant differences in taste profiles between samples. Sweetness values for different samples were shown in Figure 33B. 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 33 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 34, 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 34 The interaction between the sweet taste receptor and TLP-A

As shown in Figure 35, 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 35 The interaction between the sweet taste receptor and TLP-B