An Endogenous, Flavor-Enhancing TRV/Agrobacterium System for Edible Tomato Fruits with the Sweet Protein Thaumatin II

Abstract

The rise in diabetes and obesity worldwide has created an urgent demand for low-sugar, nutrient-dense foods with appealing flavors. This study established an endogenous and “rapid validation–stable production” platform to enhance the flavor of edible tomato fruits by integrating two key technologies in the MicroTom cherry tomato: (1) TRV viral vector-mediated transient expression and (2) Agrobacterium-mediated stable genetic transformation. We employed the human sweet taste receptor TAS1R2 for in vitro functional validation and objectively demonstrated that tomato-derived recombinant thaumatin II exhibits receptor-binding activity equivalent to that of the native protein, overcoming the limitations of traditional sensory evaluation. Non-targeted metabolomic analysis (covering 1236 metabolites) confirmed that thaumatin II expression did not significantly alter the profiles of sugars, organic acids, or key flavor compounds in tomato fruits. This provides safety data supporting the development of “ready-to-eat sugar-substitute fruits.” Our strategy offers a solution and theoretical technical support for the development of low-sugar, high-nutrient foods.

1. Introduction

Over the past two decades, excessive sugar consumption has emerged as a major contributor to the global surge in diabetes, cardiovascular diseases, and obesity [1,2]. Since the World Health Organization (WHO) issued its 2015 guidelines limiting free sugar intake, the demand for healthy, low-calorie, sugar-free foods has grown dramatically, with “sugar-free” becoming a key label in health-conscious diets [3]. Although artificial non-nutritive sweeteners (e.g., aspartame and sucralose) are widely used in beverages and processed foods to meet sweetness demands, growing concerns persist regarding their potential carcinogenic risks [4] and environmental pollution. In contrast, plant-derived natural sugar substitutes, particularly natural sweet-tasting proteins, have gained prominence owing to their high sweetness, ease of digestibility into amino acids, and superior safety profile, making them promising candidates for use in beverages, snacks, and functional foods [5,6,7,8,9].

Among the known plant sweet proteins (e.g., thaumatin, monellin, brazzein, curculin, mabinlin, and pentadin), thaumatin II, sourced from the West African plant Thaumatococcus daniellii (Benth.), is notable for its excellent properties (Table 1). Mature thaumatin II consists of 207 amino acids folded into three domains, stabilized by eight disulfide bonds that confer remarkable structural rigidity. Notably, Domain III, a compact disulfide-rich region (residues 54–84) containing two critical residues (Lys67 and Arg82), has been identified as a key structural determinant of sweetness perception [10]. Kaneko and Kitabatake [11] demonstrated that even under extreme conditions (pH 2.0, 80 °C for 4 h), thaumatin II retains its sweet taste, highlighting its thermostability and acid resistance, which are crucial for food processing applications. Currently, thaumatin is FDA-approved as a safe sweetener with no reported genotoxicity, teratogenicity [12], or other hazards, and is commercially utilized in products including chewing gum and chocolate [13,14].

The large-scale production of thaumatin II for use as a food additive faces significant challenges. First, the natural source T. daniellii (Benth.) has stringent growth requirements, and attempts to cultivate it ex situ have been unsuccessful worldwide [15]. Second, the extraction process inevitably results in substantial yield loss. Given these limitations, recombinant DNA technology has been employed to enable heterologous expression of sweet proteins. In 1982, Edens et al. [16] observed that Escherichia coli-derived thaumatin II predominantly formed inclusion bodies, requiring denaturation and renaturation procedures that are time-consuming and yield low recovery rates. This inefficiency is attributable to the inability of prokaryotic systems to support the unique structure of plant-derived thaumatin II, which contains eight intramolecular disulfide bonds [17]. Alternative expression platforms, including Bacillus subtilis, Streptomyces lividans [18], Aspergillus awamori [19], and Pichia pastoris [20] have been used; however, critical bottlenecks persist, including inconsistent expression levels, low secretion efficiency, difficulties in optimizing process parameters [20], and complex purification protocols, all of which severely hinder commercial applications.

An alternative approach involves engineering crop plants as natural carriers, offering advantages such as low-cost raw materials, the capacity for complex protein folding and post-translational modifications, the potential elimination of downstream purification, and at the same time, provide a naturally sweeter-tasting food. Tomato (Solanum lycopersicum), a widely consumed fruit with a slightly tart flavor, is naturally low in sugar and has a low glycemic index. These attributes, combined with our sweetening enhancement, make tomato an ideal choice for bioengineering to achieve a sweet flavor experience while meeting the dietary needs of health-conscious individuals—particularly those who seek to manage blood sugar without compromising on taste. Two primary strategies can be employed to achieve this: transient transfection and transgenic technology. Viral vector systems have proven effective for high-level foreign protein expression and plant gene function studies [21]; however, no reports exist on the virus-mediated transient expression of thaumatin II in tomato. Furthermore, systematic evaluations of the effects of heterologous protein expression on critical nutritional components (e.g., sugars, organic acids, and flavor metabolites) in host plants remain scarce—a crucial consideration for developing “edible, low-calorie sweet fruit.”

Table 1. Comparative molecular characteristics of natural sweet proteins.

Sweet Protein	Source	Size (AA/MW)	Active Form	Variants	Sweetness Factor (Based on Weight)	Stability	Applications	References
Thaumatin	Thaumatococcus daniellii Benth.	207AA	Monomer	I, II, a, b, c (a)	3000 (licorice-like aftertaste)	-Soluble -Extreme acid/heat tolerance (80 °C, pH 2.0 for 4 h) -Aggregates > 70 °C at pH 7.0	-FDA-approved natural flavor enhancer and high-intensity sweetener -Approved in multiple countries	[10,11,13,14,22]
Monellin	Dioscoreophyllum cumminsii Diels	45AA (A chain) 50AA (B chain)	Dimer (A + B)	-	3000	-Heat-labile (>50 °C) -Low pH tolerance	-Safety not fully evaluated -Not approved as sweetener	[23,24,25,26]
Brazzein	Pentadiplandra brazzeana Baillon	54AA	Monomer	I, II, III	2000 (Sucrose-like taste profile, sweetness potency varies by isoform [27])	-Solubility: Good -Best thermostability among sweet proteins: -No property loss after 2 h at 98 °C -Stable at pH 2.5–8 (4 h at 80 °C)	-Traditionally used by indigenous peoples [28] -Potential allergenicity concerns prevent approval [7]	[29,30]
Curculin	Curculligo latifolia	114AA	Monomer	-	550 (taste-modifying)	-Good stability -Taste-modifying activity remains after 1 h at 50 °C (pH 3–11)	-Recognized as safe food additive in Japan [31] -Not approved in US/EU [32]	[33,34]
Mabinlin	Capparis masailai Levi	33AA (A chain) 72AA (B chain)	Dimer (A + B)	I, II-a, III, IV (a)	100	-High thermostability: -Mabinlin-II-a: Sweetness unchanged after 48 h at boiling point [35] -Mabinlin-III/IV (a): Sweetness unchanged after 1 h at 80 °C [36]	Not FDA-approved	[36,37]
Pentadin	Pentadiplandra brazzeana Baillon	-	Putative cross-linked brazzein analog	-	500 (synergistic with saccharin to suppress bitter aftertaste)	Stable at 100 °C for 5 h	-	[7,38,39,40]

The superscript ‘(a)’ indicates a subtype, following the original convention of the reference.

Table 1. Comparative molecular characteristics of natural sweet proteins.

Sweet Protein	Source	Size (AA/MW)	Active Form	Variants	Sweetness Factor (Based on Weight)	Stability	Applications	References
Thaumatin	Thaumatococcus daniellii Benth.	207AA	Monomer	I, II, a, b, c (a)	3000 (licorice-like aftertaste)	-Soluble -Extreme acid/heat tolerance (80 °C, pH 2.0 for 4 h) -Aggregates > 70 °C at pH 7.0	-FDA-approved natural flavor enhancer and high-intensity sweetener -Approved in multiple countries	[10,11,13,14,22]
Monellin	Dioscoreophyllum cumminsii Diels	45AA (A chain) 50AA (B chain)	Dimer (A + B)	-	3000	-Heat-labile (>50 °C) -Low pH tolerance	-Safety not fully evaluated -Not approved as sweetener	[23,24,25,26]
Brazzein	Pentadiplandra brazzeana Baillon	54AA	Monomer	I, II, III	2000 (Sucrose-like taste profile, sweetness potency varies by isoform [27])	-Solubility: Good -Best thermostability among sweet proteins: -No property loss after 2 h at 98 °C -Stable at pH 2.5–8 (4 h at 80 °C)	-Traditionally used by indigenous peoples [28] -Potential allergenicity concerns prevent approval [7]	[29,30]
Curculin	Curculligo latifolia	114AA	Monomer	-	550 (taste-modifying)	-Good stability -Taste-modifying activity remains after 1 h at 50 °C (pH 3–11)	-Recognized as safe food additive in Japan [31] -Not approved in US/EU [32]	[33,34]
Mabinlin	Capparis masailai Levi	33AA (A chain) 72AA (B chain)	Dimer (A + B)	I, II-a, III, IV (a)	100	-High thermostability: -Mabinlin-II-a: Sweetness unchanged after 48 h at boiling point [35] -Mabinlin-III/IV (a): Sweetness unchanged after 1 h at 80 °C [36]	Not FDA-approved	[36,37]
Pentadin	Pentadiplandra brazzeana Baillon	-	Putative cross-linked brazzein analog	-	500 (synergistic with saccharin to suppress bitter aftertaste)	Stable at 100 °C for 5 h	-	[7,38,39,40]

The superscript ‘(a)’ indicates a subtype, following the original convention of the reference.

Transgenic crops play a pivotal role in bioreactor systems because of their mature protocols and stable inheritance. Firsov et al. [41] demonstrated that transgenic tomatoes can serve as effective biofactories for recombinant thaumatin (achieved recombinant thaumatin accumulation of 50 mg/kg fresh weight in transgenic tomatoes), capable of producing the protein in a functionally intact form. This work lays a solid foundation that the transgenic tomatoes can be engineered to accumulate recombinant thaumatin, functioning as a natural source of sweetness. It suggests that transgenic tomatoes could serve as thaumatin producers, potentially revolutionizing food industry applications by enabling direct consumption of sweet, low-sugar fruits and vegetables by diabetics and health-conscious consumers, moving beyond the mere additive use of microbial-extracted products. However, the current functional validation relies predominantly on subjective taste panels rather than biological assays. Although electronic tongues/noses have been developed for small-molecule sweeteners, their application in recombinant sweet protein detection remains unreported.

To address these critical gaps, we propose a strategy for thaumatin II production. Our innovation integrates a TRV viral vector-mediated transient system (see Supplementary Information, SI) with Agrobacterium-mediated stable transformation in a cherry tomato model (S. lycopersicum cv. MicroTom), establishing an efficient system combining two well-established technologies for rapid validation and stable production of thaumatin II. For functional assessment, we conducted the first reported in vitro human sweet taste receptor (TAS1R2) interaction analysis to objectively evaluate the sweetness activity of recombinant thaumatin II, thereby overcoming the limitations of sensory evaluation. Concurrently, untargeted metabolomic profiling of 1236 metabolites was used to systematically evaluate the impact on core nutritional and flavor components, providing critical data for developing safe, edible “sweetener fruits.” This approach demonstrates the feasibility of tomatoes as thaumatin II natural carriers, enabling direct protein consumption while bypassing costly, inefficient purification processes, drastically reducing production costs and environmental burdens to deliver a transformative solution for low-sugar, healthy food industries.

2. Materials and Methods

2.1. Plasmid Construction

The pTRV2 vector used in this study was designed by our laboratory (Shenzhen University, College of Life Sciences and Oceanography) for transient expression of foreign genes in Solanaceae crops and maintained in a −20 °C freezer. It is a shuttle vector containing a kanamycin resistance gene along with the right and left border sequences of the Agrobacterium tumefaciens Ti plasmid. This facilitates the transfer of target genes into tomato leaves via Agrobacterium-mediated transformation. The T-DNA fragments included the CaMV 35S promoter, TRV viral CP protein, and target gene expression cassette. The preprothaumatin II gene (derived from T. daniellii), fused to a C-terminal FLAG tag, was cloned into the expression frame and replicated along with the viral genome. The full sequence and vector map are provided in the Supplementary Information.

Another vector, pBWA(V)HS, was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). It contains the right and left border sequences of the A. tumefaciens Ti plasmid, as well as a HygR resistance gene driven by the enhanced CaMV 35S promoter. Successfully transformed explants grew normally in HygR-containing media. The preprothaumatin II gene was fused to a 3 × HA tag at the C-terminus for detection and purification. Oligonucleotides (synthesized using GenScript) were cloned downstream of the CaMV 35S promoter and linked to the NOS terminator.

Two constructs, TRV2_CaMV35S_Thaumatin II_FLAG (Figure 1A) and pBWA(V)HS_CaMV35S_Thaumatin II_3 × HA, were generated and transformed into Agrobacterium GV3101 (Beyotime). Positive colonies were selected based on their resistance to kanamycin and hygromycin. In addition, pTRV1, a component of viral vector replication, was constructed and transformed into Agrobacterium GV3101. TRV2_CaMV35S_mCherry was used as a control for TRV2-mediated transformation, and wild-type MicroTom served as a control for transgenic experiments.

2.2. Plant Materials

Cherry tomato seeds (S. lycopersicum cv. MicroTom) were provided by the Epigenetics Research Laboratory of Shenzhen University, China. Agrobacterium cells harboring the successfully transformed TRV2_CaMV35S_Thaumatin II_FLAG plasmid were collected and resuspended in infiltration buffer (MMA: 10 mmol/L MgCl₂, 10 mmol/L MES, and 100 mmol/L AS). The bacterial suspension was adjusted to OD₆₀₀ = 0.6 using a UV spectrophotometer and incubated at 25 °C for 3 h to induce gene expression. A 1 mL syringe was used to inject Agrobacterium cultures containing pTRV1 and pTRV2_CaMV35S_Thaumatin II_FLAG into the lower leaves of four-leaf-stage plants. TRV2_CaMV35S_mCherry-transfected plants served as controls.

An Agrobacterium-mediated leaf explant transformation method was used for transgenic tomato plants. Cotyledons from two-week-old MicroTom seedlings were excised and inoculated onto callus-inducing MS medium (MS + 0.5 mg/L IAA + 1.0 mg/L 6-BA + 3% sucrose + 0.7% agar) and incubated in the dark at 25 °C to induce callus formation. An Agrobacterium suspension harboring pBWA(V)HS_CaMV35S_Thaumatin II was inoculated onto tomato tissue blocks overnight. Subsequently, the calli were transferred to hygromycin-supplemented medium for regenerant selection. Differentiated calli were then transferred to a differentiation medium (MS + 0.02–0.05 mg/L IAA + 1.0 mg/L 6-BA + 3% sucrose + 0.7% agar) and cultured under light at 25 °C. When shoots reached 2–3 cm in height, they were transferred to the rooting medium and cultured for an additional 10–15 days. Finally, the acclimatized plants were transplanted into soil. Wild-type MicroTom plants were used as controls.

2.3. RNA Extraction and RT-PCR Analysis

The tomato leaf tissue samples were frozen in liquid nitrogen and ground using a tissue grinder. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform extraction (1:5 v/v). After a 5 min incubation, the mixture was centrifuged at 12,000 rpm and 4 °C for 15 min. The RNA-containing supernatant was precipitated with isopropanol, washed with 75% ethanol, and dissolved in diethyl pyrocarbonate-treated water. cDNA was synthesized using the One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Carlsbad, CA, USA) with random hexamer primers under the following conditions: 25 °C for 10 min, 42 °C for 15 min, and hold at 4 °C.

Gene-specific primers (Sangon Biotech, Shanghai, China) were used for PCR amplification.

For TRV2_CaMV35S_Thaumatin II:

Forward: 5′-TGGTAGCATTTGAGTTTCGCA-3′

Reverse: 5′-GGGACATGCCCGGGCCTCGAG-3′

For pBWA(V)HS_CaMV35S_Thaumatin II:

Forward: 5′-TCAACCAGTACGGCAAGGAC-3′

Reverse: 5′-CAAGGGCAGTAGGGCAGAAA-3′

PCR conditions: 98 °C for 3 min; 35 cycles of 98 °C for 20 s, 56 °C for 20 s, and 72 °C for 40 s; followed by 72 °C for 5 min and a hold at 16 °C. The PCR products were separated on a 1.2% agarose gel and visualized under UV light.

2.4. Protein Extraction and Detection

For total soluble protein extraction, tomato leaf and fruit samples were ground in liquid nitrogen. The resulting powder was resuspended in extraction buffer (Tris-HCl, pH 8.0, EDTA, β-mercaptoethanol, protease inhibitor, and glycerol). After 30 min of incubation at 4 °C, the suspension was centrifuged at 12,000× g and 4 °C for 20 min. The supernatants were collected for further analysis. Proteins were separated using 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue G250 (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China). For Western blotting, proteins were transferred to a nitrocellulose membrane, blocked with 5% BSA at 37 °C for 2 h, and probed with mouse anti-HA monoclonal antibody (1:2000, Abcam, Cambridge, UK) at 4 °C overnight. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:4000, Abcam), and proteins were detected using an ECL kit (Beyotime, P0018FM, Shanghai, China). Controls included mCherry-transformed plants for the viral infection experiments and wild-type MicroTom for the transgenic experiments.

2.5. Thaumatin Purification and Co-Immunoprecipitation

Thaumatin was purified using an anti-HA-tagged magnetic bead kit (Beyotime, P2185S). Briefly, ground samples were lysed in buffer containing protease inhibitors, and the homogenate was centrifuged at 14,000× g and 4 °C for 5 min. The supernatant was concentrated using a 10 kDa ultrafiltration tube (Beyotime, FUF051). HA-tagged thaumatin was captured using magnetic beads and eluted for further analysis. To assess sweetness, purified thaumatin was incubated with the human sweet taste receptor protein TAS1R2. After washing, the coimmunoprecipitated products were eluted with SDS-PAGE buffer and verified by Western blot.

2.6. Tomato Metabolomic Analysis

Metabolomic profiling was performed by Wuhan MetWare Biotechnology Co., Ltd. (Wuhan, China) [42], using a widely targeted metabolomics approach. The freeze-dried tomato tissues were ground using zirconia beads at 30 Hz (MM 400; Retsch, Haan, Germany). A total of 100 mg of powder was extracted with 1.0 mL of 70% methanol, sonicated for 30 min, and incubated overnight at 4 °C. After centrifugation at 12,500× g for 10 min, the supernatant was filtered (0.22 μm, SCAA-104, ANPEL, Shanghai, China [43]) for UPLC-MS/MS analysis.

2.6.1. UPLC Conditions

Samples were analyzed on a Waters ACQUITY Premier HSS T3 column (1.8 μm, 2.1 × 100 mm) at 40 °C with a flow rate of 0.30 mL/min and an injection volume of 2 μL. Mobile phases: solvent A (5 mM ammonium acetate) and solvent B (acetonitrile). Gradient: 95% A to 5% A over 10 min, held for 2 min, returned to 95% A at 0.1 min, and equilibrated for 2.9 min.

2.6.2. MS Conditions

Data were acquired in DDA mode using MS-DIAL (version 4.9) software [44]. Parameters: sheath gas flow, 320 Arb; auxiliary gas flow, 40 Arb; temperature, 350 °C; ion spray voltage, ±3400/−3200 V; and mass range, 85–1250 Da.

2.7. Statistical Analysis

Metabolomics data were normalized and unit-variance scaled prior to multivariate analyses. Unsupervised principal component analysis (PCA) was performed using the prcomp function in R (version 4.5.1) [45] following unit-variance scaling. Hierarchical clustering analysis (HCA) and Pearson correlation coefficient (PCC) calculations were performed using the ComplexHeatmap package. For differential metabolite analysis, fold changes (FC) were computed and statistical significance was assessed using Student’s t-test (two-tailed) with Benjamini–Hochberg false discovery rate (FDR) correction; metabolites with FDR < 0.05 and |log₂FC| ≥ 1 were considered significant unless otherwise stated. Volcano/FC plots show log₂FC and −log10 (p-value). All tests were two-sided; p < 0.05 was considered significant.

3. Results

3.1. Molecular Analysis of Transiently Transfected Plants with Viruses

To transiently express large amounts of thaumatin II in tomato leaves, a TRV2 plasmid carrying thaumatin II was constructed and transformed into Agrobacterium. Through kanamycin resistance screening and colony PCR analysis, the presence of an 849 bp amplified fragment of the inserted thaumatin II gene in Agrobacterium GV3101 was confirmed (Supplementary Information Figure S1). Successfully transformed Agrobacterium cells were induced and injected into four-week-old tomato leaves to obtain TRVT-35S plants. After 14 days, RNA and protein extracted from leaf tissue were subjected to RT-PCR and Western blot, respectively, to verify the transcription and expression of the thaumatin gene. As expected, an 849 bp amplified fragment was observed in the RT-PCR results (Supplementary Information Figure S1), and strong expression of a protein approximately 26.5 kDa was observed in the Western blot results (Supplementary Information Figure S1). This apparent molecular weight is higher than the theoretical mass of the unmodified polypeptide (~25.5 kDa, see GenBank accession number in Supplementary Information S2), which is consistent with the C-terminal 3 × HA tag (~3.3 kDa) included in the plasmid (see sequence in Supplementary Information S2) and the natural cleavage of its N-terminal signal peptide within the cells [39].

3.2. Metabolomic Analysis of Infected Tomato Plants

For metabolomic analysis, a widely targeted LC tandem MS (LC-MS/MS) method was used, and a total of 1236 annotated metabolites were identified in tomato fruit tissue, including 450 lipids and lipid-like molecules; 231 organic acids and their derivatives; 147 phenylpropanoids and polyketides; 121 organic heterocyclic compounds; 120 organic oxygen compounds; 60 benzene compounds; 54 nucleosides, nucleotides, and their analogs; 33 organic nitrogen compounds; 10 alkaloids and their derivatives; 4 homogeneous non-metallic compounds; 4 lignans, neolignans, and related compounds; and 2 organic sulfur compounds. Based on the quality control analysis of these metabolites, the intra-group correlation coefficient (Pearson r) was >0.998 (Figure 2A), and the coefficient of variation (CV) of 75% of the metabolites was <0.3 (Figure 2B), indicating that the data met high-quality standards. Principal Component Analysis (PCA) of the overall samples showed that the metabolic profiles of the three groups were clearly separated, with minimal intra-group variability (Figure 2C), indicating metabolic differences between the thaumatin group and the control group. Fold change (FC) analysis (Figure 2E) and functional pathway difference analysis (Figure 2D) revealed that the thaumatin II group and the control group differed significantly only in lipid and defense-related amino acid pathways, with |log₂FC| < 0.1 for soluble sugars, organic acids, and major flavor precursors, providing direct evidence of the safety of “direct-edible sugar substitute fruits.” Additionally, metabolites with relatively large fold differences included phenylbutyrylglutamine (BYGN03669, log₂FC = 22.1) and isoleucine (BYCP0082, log₂FC = 19.928), which may originate from the activation of plant defense signaling pathways triggered by thaumatin. The downregulation of polyunsaturated fatty acids may result from a burst of reactive oxygen species (ROS) induced by thaumatin, thereby reducing membrane fluidity and decreasing pathogen spread. In summary, the results indicate that virus-mediated transient transformation of the thaumatin gene does not affect the nutritional components of tomatoes but instead triggers systemic defense activation in tomato plants.

3.3. Transgenics and Regeneration

After stable transformation of explants with the same gene cassette via Agrobacterium, hygromycin-resistant calli were generated on the selection medium within 6–7 weeks, and roots differentiated from the calli after 10 weeks of culture (Figure 3A). Explants that were not successfully inoculated did not survive in the selection medium. The transformation experiment yielded 33 regenerated plant lines (designated GMOT-35S lines). All 33 transgenic tomato lines grew normally, with no observable differences in growth or morphological characteristics between transgenic and non-transgenic tomato plants. For further characterization, all hygromycin-resistant lines derived from independent transformation events were grown in a greenhouse. After seedling formation, leaf samples were collected for DNA extraction and PCR, confirming the presence of the hygromycin gene in 30 regenerated plants, except for GMOT-35S-18, GMOT-35S-19, and GMOT-35S-26 (Figure 3B).

3.4. Characterization of Transgenic Plant Thaumatin Ⅱ

To detect thaumatin II, amplification primers derived from the thaumatin II gene sequence were designed. Total RNA was extracted from leaf and fruit samples and subjected to RT-PCR. A 351 bp Thaumatin II gene fragment was amplified from the cDNA of GMOT-35S-1, GMOT-35S-2, GMOT-35S-3, GMOT-35S-5, and GMOT-35S-7 transgenic plants (Figure 4A). Among these, GMOT-35S-1, GMOT-35S-2, and GMOT-35S-3 showed clear bands and were selected for Western blot analysis. The results showed a single band corresponding to thaumatin II with a molecular weight of approximately 26.5 kDa, in both leaf and fruit samples. No comparable immunoreactive band was observed in the protein samples of the untransformed control group plants (Figure 4B). This indicates that recombinant thaumatin II was correctly expressed and folded, existing mainly as a soluble protein in tomato leaves and fruits.

To estimate the yield, the collected samples were weighed during the sampling process. The thaumatin II protein content was then determined using a thaumatin II-specific ELISA kit (Hnybio, Shanghai, China, Catalog No. HB212-SH), following a procedure that included enzyme conjugation, incubation, and washing steps. Through this we could estimate the production level, and the results indicated an approximate yield of 0.252 mg/kg. We note the yield difference compared to that of Firsov et al. [41] (50 mg/kg), which primarily stems from the difference in research objectives. Their aim was to maximize protein production, hence the use of a large-fruited, high-biomass cultivar (‘Yalf’) and a process optimized for protein extraction and purification. In contrast, we selected ‘MicroTom’ for its short growth cycle and well-characterized flavor background, making it highly suitable for rapid screening and functional characterization of flavor traits. The extraction buffer we employed was primarily designed for organelle studies, which may also have limited the complete release and quantification of the target protein.

3.5. Interaction Analysis of Recombinant Thaumatin Ⅱ-T1RS2

To explore the sweet taste characteristics of recombinant thaumatin II in a biological context, protein–protein interaction (PPI) predictions between thaumatin II and TAS1R2 and TAS1R3 were performed using AlphaFold3 [46] and HDOCK, respectively, and the key binding sites on thaumatin II that interact with the sweet taste receptor heterodimer were obtained (Figure 5 and Figure 6). The results showed that in the AlphaFold model, the binding checkpoints of thaumatin II with TAS1R2 were ASP-21, ARG-79, TYR-99, LYS-163, GLY-162, and PRO-188, and the binding checkpoints with TAS1R3 were ARG-67, LYS-139, and PRO-141. In the HDOCK model, the binding checkpoints of thaumatin II with TAS1R2 were concentrated on ARG-79, TYR-95, and THR-160, whereas binding with TAS1R3 was located at GLN-42, all within the central region of the receptor. By analyzing the binding tightness (

Table 2. Key amino acids related to docking and their binding strength with the protein, as predicted by AlphaFold.

Thaumatin II	TAS1R2	Distance (Å)	Interface Area (Å2)	ΔiG (kcal/mol)
SP-21	LYS-497	3.3	584.3	−0.7
ARG-79	CYS-517	3.2
TYR-99	ASN-292	3.0
LYS-163	GLN-237	3.7
GLY-162	LYS-263	2.1
PRO-188	ASP-262	2.9
Thaumatin II	TAS1R3	Distance (Å)	Interface Area (Å2)	ΔiG (kcal/mol)
ARG-67	ASP-544	3.2	442.1	0.1
LYS-139	GLN-531	3.0
PRO-141	BLN-531	3.0

Å(2) indicates the interface area; ΔiG indicates the solvation free energy gain upon interface formation.

and

Table 3. Key amino acids related to docking and their binding strength with the protein, as predicted by HDOCK.

Thaumatin Ⅱ	TAS1R2	Distance (Å)	Interface Area (Å2)	ΔiG (kcal/mol)
ARG-79	GLU-516	3.4	731.5	−0.1
TYR-95	ASP-711	3.1
THR-160	SER-550	2.9
Thaumatin Ⅱ	TAS1R3	Distance (Å)	Interface Area (Å2)	ΔiG (kcal/mol)
GLU-42	GLN-543	3.2	56.0	1.3

Å(2) indicates the interface area; ΔiG indicates the solvation free energy gain upon interface formation.

), we conclude that TAS1R2 and TAS1R3 participate in the perception of thaumatin II’s sweet taste in the form of a heterodimer. Among these interactions, thaumatin II exhibits a larger contact area and binding strength with TAS1R2.

Previous studies have shown that TAS1R2 can form non-functional homodimers when co-expressed with TAS1R3 in tissue culture cells, introducing significant confusion into the particle analysis. Additionally, TAS1R1 and TAS1R3 are involved in the perception of umami, whereas TAS1R2 and TAS1R3 are responsible for the perception of sweetness, indicating the functional or auxiliary properties of TAS1R3 (Figure 6A). To explore the sweetness of recombinant thaumatin II, immunoprecipitation was performed using the human receptor protein TAS1R2 and purified thaumatin II protein samples (from GMOT-35S-2 fruit). The obtained samples were subjected to Western blot analysis, and a significant band was detected between the immunoprecipitation product and purified thaumatin II at 26.5 kDa. Concurrently, the product contained His-tagged TAS1R2 at 66 kDa (Figure 6B), indicating that heterologous expression of thaumatin II can bind to TAS1R2, further confirming its sweet taste properties.

4. Discussion

By integrating the transient expression system of the TRV viral vector with Agrobacterium-mediated stable genetic transformation, an efficient system for rapid validation and stable production was established. This approach enabled rapid functional assessment of the thaumatin II gene and preliminary optimization of production parameters. Ultimately, transgenic tomato lines (T-35S) stably expressing high levels of thaumatin II (approximately 26.5 kDa) in both the leaves and fruits were obtained. This streamlined production system eliminates the need for downstream purification steps, as thaumatin II can be directly obtained from edible fruit juice. These findings provide a strong technical foundation for scalable and controllable plant-based thaumatin II production, effectively overcoming the limitations of scarce natural resources and traditional microbial fermentation methods.

Ensuring that heterologous protein expression does not compromise the nutritional or flavor quality of the host crop is a prerequisite for developing edible sugar-substituting fruits. In this study, untargeted metabolomic analysis of 1236 fruit metabolites revealed that thaumatin II expression did not significantly alter the core nutritional profile of tomato fruits, including major sugars (glucose, fructose, and sucrose), organic acids (citric acid and malic acid), and most flavor-related compounds (e.g., phenylpropanoids and heterocyclic organic compounds). These results provide safety and quality data supporting the use of tomato fruits as direct carriers of thaumatin II, providing evidence for the feasibility of the “sugar-substituting fruit” concept. Notably, metabolomic profiling of transiently expressed lines also suggested that thaumatin II expression might enhance the plant’s immune defense response by increasing the levels of glutamine [48], isoleucine [49], and polyunsaturated fatty acids, implying potential stress resistance benefits. This aligns with previous findings that transgenic strawberry plants expressing thaumatin II exhibit resistance to Botrytis cinerea [50]. Thus, metabolomic analysis may serve as a powerful tool for elucidating the disease resistance mechanisms of thaumatin II, warranting further investigation.

Considering the widespread use of sweeteners in daily life, the taste and safety of synthetic thaumatin II are critical. There is a growing market demand for high-quality, non-toxic sweeteners [51]. A lifecycle assessment of thaumatin highlighted the need to evaluate its contribution to sweetness, as its sensory properties differ from those of sugar (e.g., delayed onset of sweetness compared to sugar), and it is rarely used alone [52]. Previous studies have demonstrated that the binding of sweet proteins to TAS1R2 directly correlates with known sweetness perception mechanisms [53,54]. Molecular docking studies have shown that thaumatin II binds to the central region of the heterodimeric sweet taste receptors TAS1R2 and TAS1R3, specifically to the cysteine-rich domain (CRD), whereas sucrose, aspartame, and sucralose interact with the extracellular N-terminal domain (NTD) of TAS1R1 [55]. This study identified key binding residues (Arg79 and Tyr99) and, for the first time, employed the human sweet taste receptor TAS1R2 for in vitro functional evaluation of plant-derived recombinant thaumatin II. Rather than relying solely on subjective sensory evaluations, our study adopted a receptor-based molecular approach, which provides direct and reproducible evidence of sweetness activity at the biological level. Co-immunoprecipitation experiments confirmed that thaumatin II purified from transgenic tomatoes specifically bound to TAS1R2. This objective biological validation provides reliable scientific evidence that recombinant thaumatin II exhibits sweetness activity comparable to that of the native protein, overcoming the limitations of human sensory tests and conventional sweetness evaluation methods (e.g., concentration-based assessments) [56]. Thus, this approach offers a novel strategy for detecting sweetness. Nevertheless, we acknowledge that receptor binding cannot fully substitute for in vivo taste perception, and future work will integrate sensory panel evaluations to complement the molecular findings.

Notably, the present study primarily focused on the establishment and functional validation of the transformation system and the initial characterization of T0 plants. While we have successfully demonstrated proper expression and correct processing of thaumatin II in T0 plants through molecular and functional assays, the analysis of transgene stability and inheritance patterns in subsequent generations remains an essential next step. Additionally, untargeted metabolomics, while comprehensive, may overlook subtle relevant metabolic changes and more definite evidence of causal relationships between the transgene and observed metabolic shifts is needed. Future work will involve cultivating the T1 progeny from the selected homozygous T0 lines to perform segregation analysis and assess the inheritance of the transgene. This will be crucial for confirming the development of a true-breeding, stable transgenic line suitable for scalable agricultural applications. Furthermore, targeted metabolomics is needed to further characterize the system.

The tomato-based plant expression system developed in this study represents a paradigm shift in thaumatin II production. Its core advantage lies in enabling consumers to ingest functional thaumatin II directly by eating tomato fruits, bypassing the costly, complex, and protein-loss-prone downstream purification processes associated with traditional extraction and microbial fermentation methods. This significantly reduces production costs and minimizes chemical use and waste generation. The cherry tomato cultivar MicroTom, with its compact fruit size, short growth cycle, and well-established genetic manipulation system, is an ideal chassis. This strategy can be extended to other Solanaceae crops (e.g., common tomatoes and peppers) as well as berry fruits, offering diverse “sweet yet low-/no-sugar” dietary options for diabetics, obese individuals, and health-conscious consumers. This approach transcends the current limitation of adding purified sweet proteins only to processed foods, enabling comprehensive “sugar-free” dietary solutions.

5. Conclusions

Our results indicate that a TRV/Agrobacterium dual-route transformation approach can be effectively applied to MicroTom tomato for the production of the sweet protein thaumatin II. This system provides a new pathway for low-cost, sustainable thaumatin II production and represents an advancement in healthy sweetener applications through its “sugar-substituting fruit” format. This approach demonstrates promising potential for applications in the health food industry. Future studies will be necessary to evaluate long-term transgene stability over multiple generations and to assess agronomic performance through field trials under practical growing conditions. Further research may also explore tissue-specific expression strategies to enhance production efficiency and reduce metabolic burden.